AMP-activated protein kinase supports the NGF-induced viability of human HeLa cells to glucose starvation.
ABSTRACT As an important cellular energy regulation kinase, AMP-activated protein kinase (AMPK) has been demonstrated as a key molecule in the development of tolerance to nutrient starvation. Activation of AMPK includes the phosphorylation of Thr172 of the alpha-subunit. Nerve growth factor (NGF) was originally isolated for its ability to stimulate both survival and differentiation in peripheral neurons, but many investigations have shown that the NGF also plays an important role in survival, growth and invasion of many human cancers. In this study, we used CCK-8 cell viability assay to find that NGF could facilitate the viability of HeLa cells following glucose deprivation while not in glucose-normal control groups. This effect of NGF-induced viability promotion to glucose starvation can be suppressed by Compound C, a specific inhibitor of AMPK. Meanwhile, western blot analysis showed that AMPKalpha1/alpha2 Thr172 phosphorylation level in HeLa cells was up-regulated after NGF treatment under glucose starvation, and Compound C was able to reduce the AMPKalpha1/alpha2 Thr172 phosphorylation level which was up-regulated by NGF in HeLa cells. Taken together, these results indicate that AMP-activated protein kinase supports the NGF-induced viability of human HeLa cells to glucose starvation.
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ABSTRACT: The distal cytoplasmic motifs of leukemia inhibitory factor receptor α-chain (LIFRα-CT3) can independently induce intracellular myeloid differentiation in acute myeloid leukemia (AML) cells by gene transfection; however, there are significant limitations in the potential clinical use of these motifs due to liposome-derived genetic modifications. To produce a potentially therapeutic LIFRα-CT3 with cell-permeable activity, we constructed a eukaryotic expression pcDNA3.0-TAT-CT3-cMyc plasmid with a signal peptide (ss) inserted into the N-terminal that codes for an ss-TAT-CT3-cMyc fusion protein. The stable transfection of Chinese hamster ovary (CHO) cells via this vector and subsequent selection by Geneticin resulted in cell lines that express and secrete TAT-CT3-cMyc. The spent medium of pcDNA3.0-TAT-CT3-cMyc-transfected CHO cells could be purified using a cMyc-epitope-tag agarose affinity chromatography column and could be detected via SDS-PAGE, with antibodies against cMyc-tag. The direct administration of TAT-CT3-cMyc to HL-60 cell culture media caused the enrichment of CT3-cMyc in the cytoplasm and nucleus within 30 min and led to a significant reduction of viable cells (P < 0.05) 8 h after exposure. The advantages of using this mammalian expression system include the ease of generating TAT fusion proteins that are adequately transcripted and the potential for a sustained production of such proteins in vitro for future AML therapy.Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas / Sociedade Brasileira de Biofisica ... [et al.] 06/2012; 45(10):913-20. DOI:10.1590/S0100-879X2012007500101 · 1.08 Impact Factor
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ABSTRACT: The evolutionarily conserved serine/threonine kinase, AMP-activated protein kinase (AMPK), functions as a cellular fuel gauge that regulates metabolic pathways in glucose and fatty acid metabolism and protein synthesis, and recent data demonstrate that it also plays a critical role in systemic energy balance. PRKAA2, the gene that encodes the α2 catalytic subunit of AMPK, showed be involved in the glucose and lipid metabolism. To date, genetic variants in human PRKAA2 have been shown associations with type 2 diabetes (T2D) in several populations, but few studies show a complete description of the variability of bovine PRKAA2. In the present study, we reported the investigation of PRKAA2 genetic polymorphisms in three Chinese indigenous bovine breeds [Qinchuan (n = 328), Nanyang (n = 278), Jiaxian (n = 148)] and yak (n = 57). The screening of all exons including adjacent splice sites of the gene was performed using a PCR-SSCP strategy, and following sequence analysis revealed fifteen single nucleotide polymorphisms (SNPs). Five SNPs were identified in exons, which all generate synonymous mutations, and other identified variations were located in introns. Linkage disequilibrium (LD) coefficients and haplotype frequencies for some SNPs were investigated. In total, six haplotypes were found in the cattle breeds. Two LD blocks were found in Qinchuan cattle and three common haplotypes were identified based on four SNPs, with the most common haplotype (TGCT) occurring at a frequency of 53.7%; three of the four possible haplotypes were found in Nanyang cattle, with the most common haplotype (CT) occurring at a frequency of 56.3%; whereas, no suitable haplotypes were found in Jiaxian cattle and yak. Phylogenetic analysis showed that Qinchuan and Jiaxian were firstly clustered together, and then Nanyang was added to the branch. The yak (Poephagus grunniens) diverged strongly from the branch of the Bos Taurus. These data will provide a background for more extensive characterization of the bovine PRKAA2 gene, its diversity in different cattle breeds, and evolutionary information of Chinese cattle breeds.Molecular Biology Reports 03/2011; 38(3):1551-6. DOI:10.1007/s11033-010-0263-3 · 1.96 Impact Factor
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ABSTRACT: IntroductionMatrix-detachment triggers anoikis, a form of apoptosis, in most normal epithelial cells, while acquisition of anoikis resistance is a prime requisite for solid tumor growth. Interestingly, recent studies have revealed that a small population of normal human mammary epithelial cells (HMECs) survive in suspension and generate multicellular spheroids termed as `mammospheres¿. Therefore, understanding how normal HMECs overcome anoikis may provide insights into breast cancer initiation and progression.Methods Primary breast tissue-derived normal HMECs were grown as adherent monolayers or mammospheres. The status of AMP-activated protein kinase (AMPK) and PEA15 signaling was investigated by immunoblotting. Pharmacological agents and RNA interference (RNAi) approach were employed to gauge their roles in mammosphere formation. Immunoprecipitation and in vitro kinase assays were undertaken to evaluate interactions between AMPK and PEA15. In vitro sphere formation and tumor xenograft assays were performed to understand their roles in tumorigenicity.ResultsIn this study we show that mammosphere formation by normal HMECs is accompanied with an increase in AMPK activity. Inhibition or knockdown of AMPK impaired mammosphere formation. Concomitant with AMPK activation, we detected increased Ser116 phosphorylation of PEA15, which promotes its anti-apoptotic functions. Inhibition or knockdown of AMPK impaired PEA15 Ser116 phosphorylation and increased apoptosis. Knockdown of PEA15, or overexpression of the non-phosphorylatable S116A mutant of PEA15, also abrogated mammosphere formation. We further demonstrate that AMPK directly interacts with and phosphorylates PEA15 at Ser116 residue, thus identifying PEA15 as a novel AMPK substrate. Together, these data revealed that AMPK activation facilitates mammosphere formation by inhibition of apoptosis, at least in part, through Ser116 phosphorylation of PEA15. Since anoikis resistance plays a critical role in solid tumor growth, we investigated the relevance of these findings in the context of breast cancer. Significantly, we show that the AMPK-PEA15 axis plays an important role in the anchorage-independent growth of breast cancer cells both in vitro and in vivo.Conclusions Our study identifies a novel AMPK-PEA15 signaling axis in the anchorage-independent growth of both normal and cancerous mammary epithelial cells, suggesting that breast cancer cells may employ mechanisms of anoikis resistance already inherent within a subset of normal HMECs. Thus, targeting the AMPK-PEA15 axis might prevent breast cancer dissemination and metastasis.Breast cancer research: BCR 08/2014; 16(4):420. DOI:10.1186/s13058-014-0420-z · 5.88 Impact Factor