Double hit of <i>NEMO</i> gene in preeclampsia

<div><p>The precise etiology of preeclampsia is unknown. Family studies indicate that both genetic and environmental factors influence its development. One of these factors is NFkB, whose activation depends on NEMO (NFkB essential modulator. This is the first study to investigate the association between the existence of single nucleotide variant of the <i>NEMO</i> gene and the appearance of preeclampsia. A total of 151 women (72 preeclamptic women and 79 controls) and their children were examined. Sanger sequencing was performed to identify variants in the <i>NEMO</i> gene in the preeclamptic mothers. The maternal identified variants were then sought in the studied groups of children, and in the maternal and child controls, using RFLP-PCR. Real-time RT-PCR was performed to assess <i>NEMO</i> gene expression in maternal blood, umbilical cord blood and placentas. The sequencing process indicated the existence of two different variants in the 3'UTR region of the <i>NEMO</i> gene of preeclamptic women (IKBKG:c.*368C>A and IKBKG:c.*402C>T). The simultaneous occurrence of the TT genotype in the mother and the TT genotype in the daughter or a T allele in the son increased the risk of preeclampsia development 2.59 fold. Additionally, we found that the configuration of maternal/fetal genotypes (maternal TT/ daughter TT or maternal TT/son T) of IKBKG:c.*402C/T variant is associated with the level of NEMO gene expression. Our results showed that, the simultaneous occurrence of the maternal TT genotype (IKBKG:c.*402C>T variants) and TT genotype in the daughter or T allele in the son correlates with the level of NEMO gene expression and increases the risk of preeclampsia development. Our observations may offer a new insight into the genetic etiology and pathogenesis of preeclampsia.</p></div

The influence of IKBKG:c.*402C/T variation on the secondary structure of 1A, 1B and 1C transcripts.

<p>Analysis was conducted using RNAfold software. (A) transcript 1A with 402T variant; (B) transcript 1A with 402C variant; (C) transcript 1B with 402T variant; (D) transcript 1B 402C variant; (E) transcript 1C with 402T variant; (F) transcript 1C with 402C variant. MFE—Minimum Free Energy.</p

EGFR Activation Leads to Cell Death Independent of PI3K/AKT/mTOR in an AD293 Cell Line

<div><p>The Epidermal Growth Factor Receptor (EGFR) and its mutations contribute in various ways to tumorigenesis and biology of human cancers. They are associated with tumor proliferation, progression, drug resistance and the process of apoptosis. There are also reports that overexpression and activation of wild-type EGFR may lead to cell apoptosis. To study this phenomenon, we overexpressed in an AD293 cell line two most frequently observed forms of the EGFR receptor: wild-type and the constitutively active mutant–EGFR variant III (EGFRvIII). Then, we compared the effect of EGF stimulation on cell viability and downstream EGFR signaling. AD293 cells overexpressing wild-type EGFR, despite a significant proliferation increase in serum supplemented medium, underwent apoptosis after EGF stimulation in serum free conditions. EGFRvIII expressing cells, however, were unaffected by either serum starvation or EGF treatment. The effect of EGF was completely neutralized by tyrosine kinase inhibitors (TKIs), indicating the specificity of this observation. Moreover, apoptosis was not prevented by inhibiting EGFR downstream proteins (PI3K, AKT and mTOR). Here we showed another EGFR function, dependent on environmental factors, which could be employed in therapy and drug design. We also proposed a new tool for EGFR inhibitor analysis.</p></div

Primers used in sequencing processes.

<p>Primers used in sequencing processes.</p

Comparison of clinical data within the study population.

<p>Comparison of clinical data within the study population.</p

Primer sequences.

<p>Primer sequences.</p

Serum starved EGF-treated cells expressing wild-type EGFR undergo apoptosis, what is prevented by erlotinib.

<p>(A, B) AD293par cells survive EGF treatment in contrast to massively detaching cells overexpressing EGFRwt. Medium was changed to serum free, DMSO/EGF/EGF+erlotinib was added and cells were photographed every 6 hours. Photos present cells at time 0 and at 48 hour. (C) Most of AD293 cells with wild-type EGFR treated with EGF become apoptotic. Medium was changed to serum free, DMSO/EGF/EGF+erlotinib/erlotinib was added, cells were harvested after 48 hours, stained with Annexin V FITC/propidium iodide and analyzed by flow cytometry. (D) Analysis of compacted state of chromatin in apoptotic cells confirmed the link between EGF activated EGFRwt and apoptosis. Medium was changed to serum free, DMSO/EGF/EGF+erlotinib/erlotinib was added, cells were stained after 24 hours with propidium iodide and Hoechst 33342 and analyzed under fluorescence microscopy. <i>Blue arrow</i>: <i>viable cell; green arrow</i>: <i>early apoptotic cell; yellow arrow</i>: <i>late apoptotic cell; red arrow</i>: <i>necrotic cell</i>.</p