employing-digital-droplet-pcr-to-detect-braf-v600e-mutations-formalin

Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines

ddPCR is a highly sensitive PCR method that utilizes a water-oil emulsion system. Using a droplet generator, an extracted nucleic acid sample is ...

Seoul National University

To investigate 1-cys peroxiredoxin (1-cysPrx) expression during the corneal wound-healing process after PRK and the effect of growth factors on 1-cysPrx expression in cultured bovine keratocytes (BKs).

Regulation of 1-cys peroxiredoxin expression in the process of stromal wound healing after photorefractive keratectomy.

1-cys peroxiredoxin (1-cysPrx), a member of the peroxiredoxin family with a single conserved cysteine residue, reduces a broad spectrum of hydroperoxides. This study was undertaken to examine changes in 1-cysPrx expression in human cataract samples, human lens epithelial (HLE B3) cell line, and rat organ-cultured lenses in response to oxidative insult induced by H2O2 or transforming growth factor-beta1 (TGF-beta1). Expression of 1-cysPrx mRNA and protein in HLE B3 cells increased in response to 2-8 ng ml(-1) TGF-beta1 and 50-75 microm H2O2 and then decreased below the control level at high doses (10 ng ml(-1) TGF-beta1 and 100-150 microm H2O2), as determined by Northern blot and immunoblot analysis. This reduction coincided with the decrease of cell viability. Immunoreactive 1-cysPrx protein was measured in capsulorrhexis specimens obtained from patients with anterior subcapsular cataract (ASC), nuclear sclerosis (NS), cortical spokes (CS), posterior subcapsular cataract (PSC), or white mature cataract (WC) at the time of cataract surgery. Significant reduction of 1-cysPrx protein was observed in ASC, PSC, and WC samples, but there was no statistical difference in CS and NS samples relative to normal control. Also, rat lens explants were cultured with 10 ng ml(-1) TGF-beta1 for approximately 5 days or 500 microm H2O2 for approximately 2 days. Subsequently, expression of 1-cysPrx mRNA and protein in the lens capsules was evaluated. Rat lens explants treated with TGF-beta1 or H2O2 developed a cataract similar to human ASC or WC, respectively, which resulted in a markedly decreased expression of 1-cysPrx mRNA and protein. Collectively, these findings show that expression patterns of 1-cysPrx gene in the lens are changed in response to oxidative stress, a major factor in the etiology of cataract.

Reduced expression of 1-cys peroxiredoxin in oxidative stress-induced cataracts.

To compare corneal flaps made with a femtosecond (FS) laser with those made with a mechanical microkeratome (MM) in rabbits, measuring early postoperative inflammation and apoptosis and late postoperative adhesion strength.

A femtosecond laser creates a stronger flap than a mechanical microkeratome.

To study the regulation by mitomycin C (MMC) of 1-cys peroxiredoxin (Prx) expression during the corneal wound-healing process and its induction pathway in cultured bovine keratocytes (BKs).

Expression of 1-cys peroxiredoxin in the corneal wound-healing process.

5-Aminoimidazole-4-carboxamide-ribonucleoside (AICAR) is an activator of 5'-AMP-activated protein kinase (AMPK), which plays a role in the maintenance of cellular energy homeostasis. Activated AMPK inhibits the protein kinase mechanistic target of rapamycin, thereby reducing the extent of protein translation and suppressing both cell growth and cell cycle entry. Recent reports indicate that AMPK-mediated growth inhibition is achieved via an action of the RAF-MEK-ERK mitogen-activated protein kinase pathway in melanoma cells harboring the V600E mutant form of the BRAF oncogene. In this study, we investigated the anti-cancer efficacy of AICAR by measuring its effects on proliferation, apoptosis, and cell cycle progression of BRAF wild-type and V600E-mutant thyroid cancer cell lines. We also explored the mechanism underlying these effects. AICAR inhibited the proliferation of BRAF V600E-mutant thyroid cancer cell lines more strongly than was the case with wild-type cell lines. The suppressive effect of AICAR on cell proliferation was associated with increased S-phase cell cycle arrest and apoptosis. Interestingly, AICAR suppressed phosphorylation of ERK and p70S6K in BRAF V600E-mutant thyroid cancer cells, but rather increased phosphorylation in wild-type cells. Together, the results indicate that AICAR-induced AMPK activation in BRAF V600E-mutant thyroid cancer cell lines resulted in increases in apoptosis and S-phase arrest via downregulation of ERK and p70S6K activity. Thus, regulation of AMPK activity may be potentially useful as a therapy for thyroid cancer if the cancer harbors a BRAF V600E mutation.

The influence of the BRAF V600E mutation in thyroid cancer cell lines on the anticancer effects of 5-aminoimidazole-4-carboxamide-ribonucleoside.

Patients with metastatic thyroid cancers that do not uptake iodine need effective therapeutic option. Differentiation-inducing agents have been tried to restore functional expression of sodium iodide symporter (NIS) without success. Our objective was to assess the effect of alpha-lipoic acid (ALA), known as potential antioxidant, on expression of sodium iodide symporter in thyroid cancer cells.

Alpha-lipoic acid induces sodium iodide symporter expression in TPC-1 thyroid cancer cell line.

Hepatic resection is the most curative treatment option for early-stage hepatocellular carcinoma, but is associated with a high recurrence rate, which exceeds 50% at 5 years after surgery. Understanding the genetic basis of hepatocellular carcinoma at surgically curable stages may enable the identification of new molecular biomarkers that accurately identify patients in need of additional early therapeutic interventions. Whole exome sequencing and copy number analysis was performed on 231 hepatocellular carcinomas (72% with hepatitis B viral infection) that were classified as early-stage hepatocellular carcinomas, candidates for surgical resection. Recurrent mutations were validated by Sanger sequencing. Unsupervised genomic analyses identified an association between specific genetic aberrations and postoperative clinical outcomes. Recurrent somatic mutations were identified in nine genes, including TP53, CTNNB1, AXIN1, RPS6KA3, and RB1. Recurrent homozygous deletions in FAM123A, RB1, and CDKN2A, and high-copy amplifications in MYC, RSPO2, CCND1, and FGF19 were detected. Pathway analyses of these genes revealed aberrations in the p53, Wnt, PIK3/Ras, cell cycle, and chromatin remodeling pathways. RB1 mutations were significantly associated with cancer-specific and recurrence-free survival after resection (multivariate P = 0.038 and P = 0.012, respectively). FGF19 amplifications, known to activate Wnt signaling, were mutually exclusive with CTNNB1 and AXIN1 mutations, and significantly associated with cirrhosis (P = 0.017).

Genomic portrait of resectable hepatocellular carcinomas: implications of RB1 and FGF19 aberrations for patient stratification.

5'-Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a cellular energy sensor that monitors intracellular AMP/adenosine triphosphate (ATP) ratios and is a key regulator of the proliferation and survival of diverse malignant cell types. In the present study, we investigated the effect of activating AMPK by 5-aminoimidazole-4-carboxamide-ribonucleotide (AICAR) in thyroid cancer cells.

The effect of 5-aminoimidazole-4-carboxamide-ribonucleoside was mediated by p38 mitogen activated protein kinase signaling pathway in FRO thyroid cancer cells.

Hyun-Jeung Choi

The Center for Anti-Cancer CDx,
N-Bio,
The Center for Anti-Cancer CDx, N-Bio

Seoul National University

Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines

Hyun-Jeung Choi

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}The Center for Anti-Cancer CDx,N-Bio,The Center for Anti-Cancer CDx, N-Bio

Seoul National University

Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines

The Center for Anti-Cancer CDx,
N-Bio,
The Center for Anti-Cancer CDx, N-Bio