Kim M S, Chung N G, Kang M R, Yoo N J & Lee S H(2011) Histopathology58, 660–668Genetic and expressional alterations of CHD genes in gastric and colorectal cancers Aims: Chromodomain helicase DNA-binding protein (CHD) is a regulator of the chromatin remodelling process. The aim was to determine the CHD1, CHD2, CHD3, CHD4, CHD7, CHD8 and CHD9mutational status of mononucleotide repeats in gastric and colorectal cancers with microsatellite instability (MSI). Methods and Results: The repeats were determined in 28 gastric cancers (GCs) with high MSI (MSI-H), 45 GCs with low MSI (MSI-L)/stable MSI (MSS), 35 colorectal cancers (CRCs) with MSI-H and 45 CRCs with MSI-L/MSS by single-strand conformation polymorphism analysis. CHD4 and CHD8 expressionwas also examined in GCs and CRCs by immunohistochemistry. CHD1, CHD2, CHD3, CHD4, CHD7, CHD8 and CHD9 mutations were found in five, 19, three, five, seven, 10 and seven cancers, respectively. They were detected in MSI-H cancers, but not in MSI-L/MSS cancers. Loss of CHD4 expression was observed in 56.4% of the GCs and 55.7% of the CRCs, and loss of CHD8 was observed in 35.7% of the GCs and 28.6% of the CRCs. The cancers with CHD4 and CHD8 mutations showed loss of CHD4 and CHD8 expression, respectively. Conclusions: Frameshift mutation and loss of expression of CHD genes are common in GCs and CRCs with MSI-H.These alterations might contribute to cancer pathogenesis by deregulating CHD-mediated chromatin remodelling.
Activation of nuclear factor-kappa B (NF-kappaB) signaling is considered an important mechanism in the development of prostate cancers. A recent study revealed that IkappaB kinase epsilon (IKKepsilon), an activator of NF-kappaB, was overexpressed in breast cancers and acted as an oncogene. Expression of NF-kappaB members has been reported in prostate cancer tissues, but expression of IKKepsilon has not yet been studied in prostate cancers. In this study, we attempted to explore as to whether expressions of IKKepsilon and NF-kappaB members p50/105, p52/p100 and RelA are altered in prostate cancers. We analyzed the expression of IKKepsilon, p50/105, p52/p100 and RelA in 107 prostate adenocarcinoma tissues by immunohistochemistry using a tissue microarray (TMA) method. In the TMA, IKKepsilon is expressed in basal cells, but not in alveolar cells in normal prostate glands. IKKepsilon is expressed in 60.0% of prostate intraepithelial neoplasm (PIN) and 70.1% of the prostate cancers in the cytoplasm. Nuclear immunostainings of NF-kappaB members p50/105, p52/p100 and RelA, which are considered activation of NF-kappaB signaling, were observed respectively in 28.0%, 18.7% and 37.4% of the cancers. Nuclear staining was detected neither in normal alveolar cells nor in PIN. However, none of the expression of p50/105 nor p52/p100 nor RelA nor IKKepsilon was associated with pathologic characteristics, including size of the cancers, age, Gleason score and stage. The increased cytoplasmic expression of IKKepsilon as well as the increased nuclear expressions of p50/105, p52/p100 and RelA in the prostate cancers compared to normal alveolar cells suggested that overexpression of these proteins may be related to activation of the NF-kappaB pathway and might play a role in tumorigenesis of prostate cancers.
46 Background: Recently, in the treatment of non-small cell lung cancer(NSCLC) patient, immunotherapies and targeted therapies aimed at various genes have been developed. Next-Generation Sequencing(NGS) technology is currently known as the only method for detecting genetic alterations in many different genes simultaneously. However, this technology requires a substantial amount of DNA or RNA input from patients to obtain high-quality results in a clinical setting. We have developed a Droplex NSCLC Panel Test Kit based on multiplex digital PCR, which is known as ideal molecular diagnostic technique due to its simple workflow, minimal sample input, high sensitivity and specificity. In addition, it has a one-day TAT (Turn around time) and low costs compared NGS, resulting in a lower financial burden for patients. Recently, the development of non-invasive liquid biopsy diagnostics as an alternative to tissue biopsy is emerging important issue. The kit, applying a digital PCR platform, enables multiple mutation detection using plasma samples of blood. The digital PCR-based Droplex NSCLC Panel Test Kit is designed to detect over 170 key mutations in 11 genes, namely EGFR, ALK, ROS1, BRAF, MET, RET, KRAS, HER2, NTRK1, NTRK2, and NTRK3, using DNA and RNA extracted from NSCLC FFPET and plasma samples. Methods: The Droplex NSCLC Panel Test Kit consists of four Oligo Mixtures (OMs) for detecting mutations of 4 genes on DNA and two OMs for detecting 7 gene rearrangements on RNA. After extracting DNA and RNA from a single sample, cDNA synthesis from RNA is prioritized, followed by simultaneous execution of droplet generation, PCR processes, and data analysis. The kit test was based on QX600 droplet digital PCR system of Bio-rad with 6 fluorescent channels. To validate the analytical performance of the Droplex NSCLC Panel Test Kit for each type of sample, DNA and RNA samples were extracted from FFPET sections and plasma isolated from NSCLC patients. Each type of samples, contrived samples were manufactured and tested for analytical performance. Results: The results showed that DNA/RNA multiple mutations can be detected simultaneously on a 6-fluorescent channel system. The kit was highly sensitive for low-input sample in dilutions studies ranging from 5 to 20ng DNA or RNA for FFPET samples and plasma sample. The Digital PCR NSCLC panel test detected an analytical specificity of 100% and detection limit of 0.25% or higher for all genes included in the kit. Conclusions: We demonstrated that the performance of the Droplex NSCLC Panel Test is reliable and effective for the detection of above 170 clinically relevant mutations of 11 genes in FFPET and plasma samples of patients with NSCLC. Additionally, it will be necessary to evaluate the utility of the kit through further clinical studies with NSCLC patients, as well as expanding its compatibility to various digital PCR platforms.
The topoisomerase II poisoning effect of certain protoberberine alkaloids is associated with anti-cancer activity. Structure-activity relationships of protoberberine analogues substituted on the ring protons reveal that substitution at the C19 position is an important determinant of biological activity. In this study, the effects of substituent modification at the C19 position on the interaction of protoberberines with DNA are determined using UV and NMR spectroscopy. The line broadening effect on aliphatic resonances, chemical shift changes of the imino protons of HP14 upon berberine and berberrubine binding to HP14, and the rate of the exchange process between protoberberine analogs bound indicate that berberrubine binds HP14 more specifically than berberine. In addition, the free HP14 is altered by the substituent at the 19-position. UV spectra of berberrubine have shown a hypochromic effect together with a slight red shift, which are usually regarded as characteristics of DNA intercalation. These results are consistent with our previous report that the berberrubine is partially intercalated with HP14 with molar ratio 1 : 1, whereas a non-specific interaction is predominant between the berberine and HP14.
Telomere homeostasis is regulated by telomerase and a collection of associated proteins. Telomerase is, in turn, regulated by post-translational modifications of the rate-limiting catalytic subunit hTERT. Here we show that disruption of Hsp90 by geldanamycin promotes efficient ubiquitination and proteasome-mediated degradation of hTERT. Furthermore, we have used the yeast two-hybrid method to identify a novel RING finger gene ( MKRN1 ) encoding an E3 ligase that mediates ubiquitination of hTERT. Overexpression of MKRN1 in telomerase-positive cells promotes the degradation of hTERT and decreases telomerase activity and subsequently telomere length. Our data suggest that MKRN1 plays an important role in modulating telomere length homeostasis through a dynamic balance involving hTERT protein stability.
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