Browsing by Subject "AMPK"

STK11/LKB1 is a tumor suppressor gene and mutated in 18% of lung adenocarcinomas. Tumor suppressor liver kinase B1 (LKB1) is known to activate adenosine monophosphate-activated protein kinase (AMPK) and 12 AMPK-related kinases (ARKs) by phosphorylating a conserved threonine residue in their T-loop region. A number of studies focused on investigating the influence of LKB1-AMPK signaling on cancer cell proliferation. However, there is no systematic study for identifying the critical LKB1 kinase substrates in suppressing lung cancer cell growth. In this project, the LKB1-deficient lung adenocarcinoma cell line A549 cells were sequentially overexpressed with constitutively active mutants of AMPKα1, AMPKα2, MARK1, MARK2, MARK3, MARK4, NUAK1, NUAK2, SIK1, SIK2, SIK3. The overexpression status was confirmed at both genetic and protein levels by qPCR and Western blotting, correspondingly. In vitro growth assays demonstrated up to 33% reduced growth rate of A549 cells overexpressing AMPKα1, AMPKα2 and NUAK1. Furthermore, siRNA knockdown of the selected substrates in LKB1-overexpressing A549 cells significantly rescued the cell growth defect. These findings suggest, that AMPKα1, AMPKα2 and NUAK1 kinases are critical for LKB1-mediated cell growth defect in lung adenocarcinoma.

2,3,7,8-Tetrachlorodibentso-p-dioksin (TCDD) is the most potent congener of the group of dioxins. It forms as a byproduct or impurity in many chemical processes. TCDD is enriched in the food chain and people are exposed to it in small portions chronically mainly by nutrition. TCDD has many toxic impacts on humans and animals which are mediated by the cellular aryl hydrocarbon -receptor. In spite of decades of research the toxic mechanisms of TCDD are poorly known. By investigating the toxicity of TCDD with large doses in experimental animals we will gain more insight into its toxic mechanisms and into its risks to humans. An important form of acute toxicity of TCDD is the wasting syndrome in which rats eat less, lose weight and finally die or after a sublethal dose stay thinner than littermates. The mechanism by which TCDD causes the wasting syndrome is unclear. Two central enzymes that take part in the regulation of the body's energy balance are the adenosine monophosphate activated protein kinase (AMPK) and stearoyl-CoA-desaturase 1 (Scd1). Changes in the expression or the regulation of their action could well be related to the wasting syndrome. The goal of this experiment was to compare in liver the mRNA expression of AMPK, Scd1 and control genes which are widely used in quantitative PCR in dioxin-sensitive Long-Evans (L-E) and resistant Han/Wistar (H/W) rat strains after a large (lethal to the sensitive strain) TCDD dose at early phases of intoxication (days 1, 4 and 10). There was also a L-E rat group whose feed was restricted to mimic the wasting caused by TCDD. The goal of the feed restriction was to separate the possible primary effect of TCDD from a secondary effect, depletion of energy. RNA was isolated from liver samples and the cDNA was made from isolated RNA with M-MLV-derived reverse transcriptase enzyme using oligo dT and random hexamer primers. The mRNAs of Scd1-enzyme and the control genes in the liver samples were measured using real-time quantitative PCR and specific primers. At 10 days feed restriction lowered significantly the expression of AMPK mRNA in L-E-rats. At 4 and 10 days feed restriction lowered significantly also the expression of Scd1 mRNA; on day 10 the mRNA level of control rats was about 10,000-fold higher. At day 1 TCDD elevated the expression of Scd1-enzyme. TCDD did not cause a significant decrease in the expression of Scd1-enzyme on day 4 and on day 10 the decrease was significantly less, about 1/10 of the level in control rats. These findings suggest that either TCDD inhibits the strong decrease of AMPK and Scd1 caused by energy deficiency or it causes an induction of AMPK and Scd1 which in turn is countered by the weight loss caused by the wasting syndrome. Overall it seems that rats exposed to TCDD do not recognize the energy depletion and their hepatocytes do not turn on the energy sparing mode of metabolism. Feed restriction and TCDD affected clearly also the expression of the control genes GAPDH, Pgk1 and Bact which were believed to be stable. The use of control genes which are linked to the regulation of cellular energy balance is risky in long-term feed restriction experiments.

Lung cancer is one of the most common and deadliest cancers worldwide, but the mechanisms behind different types of lung cancer are still poorly understood. Non-small cell lung cancer makes up 80% of lung cancers, and some epigenetic mechanisms have been proposed for it. Epigenetic modifications are a way of influencing the expression of genes by inhibition or activation. PRC2 is an epigenetic modulator that catalyses the formation of methyl groups on histone 3 lysine 27, which is an epigenetic mark with repressive nature. PRC2 has been proposed to be downstream of AMPK, an energy sensor of the cell, which is phosphorylated by LKB1 under energy stress conditions. Inactivating mutations in LKB1 are known to cause and worsen non-small cell lung cancer, and the overexpression of EZH2, the catalytic subunit of PRC2, has similar effects. Therefore, establishing a novel downstream mechanism linking LKB1, AMPK, and PRC2 together could explain one mechanism for NSCLC tumorigenesis. Changes in metabolism are a feature of cancer cells, and this pathway could also link energy stress and cancer together. Mouse embryonic fibroblast and H358 cell lines overexpressing wild type EZH2, mutant EZH2 and GFP were generated and treated with the glycolysis inhibitor 2-deoxyglucose to study the effects of energy stress. Levels of histone methylation and phosphorylation statuses of AMPK and its downstream target ACC were assessed with Western blotting, and expression levels of potential PRC2 target genes with RT-qPCR. The study setting proved to be functional for the response of AMPK to energy stress conditions, as both AMPK and ACC were phosphorylated in the presence of 2-DG. In mouse embryonic fibroblasts, PIM1 showed different gene expression with wild type and mutant EZH2, suggesting that its activation would be regulated through the phosphorylation of the T311 site of EZH2 during energy stress. The results from histone methylation statuses did not follow the hypothesis, possibly because of the lack of specificity of detecting global H3K27me3. Other target genes besides PIM1 in MEFs did not show significant changes in expression level. Considering that the incorporation of the mutant EZH2 into PRC2 complexes was not validated, additional research would be needed to confirm or deny the explained mechanism between PRC2 and AMPK.