AMP-activated protein kinase is required for cell survival and growth in HeLa-S3 cells in vivo.

Activation of the AMP-dependent protein kinase (AMPK) is linked to cancer cell survival in a variety of cancer cell lines, particularly under conditions of stress. As a potent activator of AMPK, metformin has become a hot topic of discussion for its effect on cancer cell. Here, we report that AMPK activated by metformin promotes HeLa-S3 cell survival and growth in vivo. Our results show that metformin inhibited cell proliferation in MCF-7 cells, but not in LKB1-deficient HeLa-S3 cells. Re-expression of LKB-1 in HeLa-S3 cells restored the growth inhibitory effect of metformin, indicating a requirement for LKB-1 in metformin-induced growth inhibition. Moreover, AMPK activation exerted a protective effect in HeLa-S3 cells by relieving ER stress, modulating ER Ca(2+) storage, and finally contributing to cellular adaptation and resistance to apoptosis. Our findings identify a link between AMPK activation and cell survival in HeLa-S3 cells, which demonstrates a beneficial effect of AMPK activated by metformin in cancer cell, and suggests a discrete re-evaluation on the role of metformin/AMPK activation on tumor cell growth, proliferation, and on clinical application in cancer therapy.

The integrator complex recognizes a new double mark on the RNA polymerase II carboxyl-terminal domain.

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (pol II) comprises multiple tandem repeats of the heptapeptide Tyr(1)-Ser(2)-Pro(3)-Thr(4)-Ser(5)-Pro(6)-Ser(7). This unusual structure serves as a platform for the binding of factors required for expression of pol II-transcribed genes, including the small nuclear RNA (snRNA) gene-specific Integrator complex. The pol II CTD specifically mediates recruitment of Integrator to the promoter of snRNA genes to activate transcription and direct 3' end processing of the transcripts. Phosphorylation of the CTD and a serine in position 7 are necessary for Integrator recruitment. Here, we have further investigated the requirement of the serines in the CTD heptapeptide and their phosphorylation for Integrator binding. We show that both Ser(2) and Ser(7) of the CTD are required and that phosphorylation of these residues is necessary and sufficient for efficient binding. Using synthetic phosphopeptides, we have determined the pattern of the minimal Ser(2)/Ser(7) double phosphorylation mark required for Integrator to interact with the CTD. This novel double phosphorylation mark is a new addition to the functional repertoire of the CTD code and may be a specific signal for snRNA gene expression.

A novel functional element in the N-terminal region of Arum concinnatum alternative oxidase is indispensable for catalytic activity of the enzyme in HeLa cells.

Alternative oxidase (AOX) is a quinol-oxygen oxidoreductase, which is known to possess a dicarboxylate diiron reaction center held in structurally postulated alpha-helical bundle. However, little is known about the structural or functional features of its N-terminal region in any organism, with the exception of a regulatory cysteine residue (CysI) in angiosperm plants. Here, we show that transcripts of two AOX1 isozymes (AcoAOX1a and AcoAOX1b) are coexpressed in thermogenic appendices of Arum concinnatum, while their enzymatic activities seem to be distinct. Namely, AcoAOX1a, an abundantly expressed transcript in vivo, shows an apparent cyanide-insensitive and n-propyl gallate-sensitive respiration during ectopic expression of the protein in HeLa cells, whereas AcoAOX1b exhibits a lower transcript expression, and appears to be totally inactive as AOX at the protein level. Our functional analyses further reveal that an E83K substitution in AcoAOX1b, which is located far upstream of CysI in the N-terminal region, is the cause of this loss of function. These results suggest the presence of a naturally occurring inactive AOX homologue in thermogenic plants. Accordingly, our results further imply that the N-terminal region of the AOX protein functionally contributes to the dynamic activities of respiratory control within the mitochondria.

Quinolinate phosphoribosyl transferase, a key enzyme in de novo NAD(+) synthesis, suppresses spontaneous cell death by inhibiting overproduction of active-caspase-3.

Quinolinate phosphoribosyl transferase (QPRT) is a key enzyme in de novo NAD(+) synthesis. QPRT enzyme activity has a restricted tissue distribution, although QPRT mRNA is expressed ubiquitously. This study was designed to elucidate the functions of QPRT protein in addition to NAD(+) synthesis. QPRT was identified as a caspase-3 binding protein using double layer fluorescent zymography, but was not a substrate for caspase-3. Surface plasmon resonance analysis using recombinant proteins showed interaction of QPRT with active-caspase-3 in a dose dependent manner at 55 nM of the dissociation constant. The interaction was also confirmed by immunoprecipitation analysis of actinomycin D-treated QPRT-FLAG expressing cells using anti-FLAG-agarose. QPRT-depleted cells showed increased sensitivity to spontaneous cell death, upregulated caspase-3 activity and strong active-caspase-3 signals. Considered together, the results suggested that QPRT protein acts as an inhibitor of spontaneous cell death by suppressing overproduction of active-caspase-3.

A new nuclear protease with cathepsin L properties is present in HeLa and Caco-2 cells.

Recently many authors have reported that cathepsin L can be found in the nucleus of mammalian cells with important functions in cell-cycle progression. In previous research, we have demonstrated that a cysteine protease (SpH-protease) participates in male chromatin remodeling and in cell-cycle progression in sea urchins embryos. The gene that encodes this protease was cloned. It presents a high identity sequence with cathepsin L family. The active form associated to chromatin has a molecular weight of 60 kDa, which is higher than the active form of cathepsin L described until now, which range between 25 and 35 kDa. Another difference is that the zymogen present in sea urchin has a molecular weight of 75 and 90 kDa whereas for human procathepsin L has a molecular weight of 38-42 kDa. Based on these results and using a polyclonal antibody available in our laboratory that recognizes the active form of the 60 kDa nuclear cysteine protease of sea urchin, ortholog to human cathepsin L, we investigated the presence of this enzyme in HeLa and Caco-2 cells. We have identified a new nuclear protease, type cathepsin L, with a molecular size of 60 kDa, whose cathepsin activity increases after a partial purification by FPLC and degrade in vitro histone H1. This protease associates to the mitotic spindle during mitosis, remains in the nuclei in binuclear cells and also translocates to the cytoplasm in non-proliferative cells.

Studies on the mode of action of diphtheria toxin. IV. Specificity of the cofactor (NAD) requirement for toxin action in cell-free systems.

The ability of a number of nucleotides related to NAD to replace NAD as cofactors for inhibition by diphtheria toxin of peptide bond formation has been examined. Neither NADH nor NADP are active. Of some 14 analogues closely related structurally to NAD that have been tested, only 3-thiocarboxamide pyridine-AD is as active as NAD itself. Replacement of the 3-carboxamide group on the pyridine ring by an acetyl group, or deamination of the purine ring, resulted in derivatives with reduced activity. The results were interpreted as suggesting that NAD and certain related nucleotides are capable of specific interaction with diphtheria toxin. Using the method of equilibrium dialysis, reversible binding of 1 mole of NAD per mole of toxin has been demonstrated. Toxoid does not interact with NAD.

Studies on the mode of action of diphtheria toxin. 3. Site of toxin action in cell-free extracts.

Extracts from HeLa cells treated with excess diphtheria toxin for several hours, until all protein synthesis has been arrested, are still able to stimulate the poly U-directed incorporation of phenylalanine into polypeptides at a moderate rate. Activity may be restored to normal levels or above by addition of a soluble enzyme fraction containing transferase II. Our results are in agreement with those of Collier who has recently shown that toxin inactivates transferase II in extracts from rabbit reticulocytes. We have further demonstrated that amino acid incorporation in extracts from intoxicated HeLa cells is limited by their transferase II content whereas, in extracts from normal cells, it is the ribosomes and to a lesser extent sRNA that are limiting. We have found that only soluble transferase II is inactivated by toxin; the ribosome-bound enzyme is resistant.

Studies on the mode of action of diphtheria toxin. V. Inhibition of peptide bond formation by toxin and NAD in cell-free systems and its reversal by nicotinamide.

Inhibition of soluble transferase II activity in cell-free systems by diphtheria toxin and NAD can be prevented or reversed in the presence of a sufficient concentration of nicotinamide. Quantitative studies on inhibition of peptide bond formation in cell-free extracts by toxin and NAD have indicated that two successive reversible reactions are involved. First, toxin and NAD interact mole for mole to form a relatively dissociable complex. This toxin-NAD complex then reacts with transferase II to form an enzymatically inactive product that is but slightly dissociated. In the presence of sufficient nicotinamide, however, the latter complex can be broken down to yield active transferase II once more. Based on the above model, an equation has been derived that accurately predicts the per cent inhibition of amino acid incorporation in cell-free systems at any given toxin and NAD level. The observed inhibition appears to be independent of the sensitivity to toxin of the cell species from which the extracts were derived, and depends only on the toxin and NAD concentrations. Although the model satisfactorily explains inhibition of peptide bond formation by toxin in cell-free systems, further assumptions are needed to explain how still lower concentrations of toxin are able to arrest protein synthesis completely in the living cell.

Studies on the mode of action of diphtheria toxin. VII. Toxin-stimulated hydrolysis of nicotinamide adenine dinucleotide in mammalian cell extracts.

When diphtheria toxin and NAD are added to soluble fractions containing aminoacyl transfer enzymes isolated from rabbit reticulocytes or from HeLa cells, free nicotinamide is released and, simultaneously, an inactive ADP ribose derivative of transferase II is formed. The reaction is reversible, and in the presence of excess nicotinamide, toxin catalyzes the restoration of aminoacyl transfer activity in intoxicated preparations. In living cultures of HeLa cells, the internal NAD concentration is sufficiently high to account for the rapid conversion, catalyzed by a few toxin molecules located in the cell membrane, of the entire cell content of free transferase II to its inactive ADP ribose derivative. Completely inactive ammonium sulfate fractions containing soluble proteins isolated from cells that have been exposed for several hours to excess toxin, can be reactivated to full aminoacyl transfer activity by addition of nicotinamide together with diphtheria toxin. Transferase II appears to be a highly specific substrate for the toxin-stimulated splitting of NAD and thus far no other protein acceptor for the ADP ribose moiety has been found.

Phosphorylation of LKB1 at serine 428 by protein kinase C-zeta is required for metformin-enhanced activation of the AMP-activated protein kinase in endothelial cells.

BACKGROUND: Metformin, one of most commonly used antidiabetes drugs, is reported to exert its therapeutic effects by activating AMP-activated protein kinase (AMPK); however, the mechanism by which metformin activates AMPK is poorly defined. The objective of the present study was to determine how metformin activates AMPK in endothelial cells. METHODS AND RESULTS: Exposure of human umbilical vein endothelial cells or bovine aortic endothelial cells to metformin significantly increased AMPK activity and the phosphorylation of both AMPK at Thr172 and LKB1 at Ser428, an AMPK kinase, which was paralleled by increased activation of protein kinase C (PKC)-zeta, as evidenced by increased activity, phosphorylation (Thr410/403), and nuclear translocation of PKC-zeta. Consistently, either pharmacological or genetic inhibition of PKC-zeta ablated metformin-enhanced phosphorylation of both AMPK-Thr172 and LKB1-Ser428, suggesting that PKC-zeta might act as an upstream kinase for LKB1. Furthermore, adenoviral overexpression of LKB1 kinase-dead mutants abolished but LKB1 wild-type overexpression enhanced the effects of metformin on AMPK in bovine aortic endothelial cells. In addition, metformin increased the phosphorylation and nuclear export of LKB1 into the cytosols as well as the association of AMPK with LKB1 in bovine aortic endothelial cells. Similarly, overexpression of LKB1 wild-type but not LKB1 S428A mutants (serine replaced by alanine) restored the effects of metformin on AMPK in LKB1-deficient HeLa-S3 cells, suggesting that Ser428 phosphorylation of LKB1 is required for metformin-enhanced AMPK activation. Moreover, LKB1 S428A, like kinase-dead LKB1 D194A, abolished metformin-enhanced LKB1 translocation as well as the association of LKB1 with AMPK in HeLa-S3 cells. Finally, inhibition of PKC-zeta abolished metformin-enhanced coimmunoprecipitation of LKB1 with both AMPKalpha1 and AMPKalpha2. CONCLUSIONS: We conclude that PKC-zeta phosphorylates LKB1 at Ser428, resulting in LKB1 nuclear export and hence AMPK activation.

DEVD-NucView488: a novel class of enzyme substrates for real-time detection of caspase-3 activity in live cells.

Live-cell detection of intracellular enzyme activity requires that substrates are cell-permeable and that the generated products are easily detected and retained in cells. Our objective was to create a novel fluorogenic substrate that could be used for real-time detection of apoptosis in living cells. We have synthesized a highly cell-permeable caspase-3 substrate, DEVD-NucView488, by linking a fluorogenic DNA-binding dye to the caspase-3 recognition sequence that renders the dye nonfunctional. On substrate cleavage, the dye is released and becomes highly fluorescent on binding to DNA. DEVD-NucView488 detected caspase-3 activation within a live-cell population much earlier and with higher sensitivity compared with other apoptosis reagents that are currently available. Furthermore, cells incubated with DEVD-NucView488 exhibited no toxicity and normal apoptotic progression. DEVD-NucView488 is an ideal substrate for kinetic studies of caspase-3 activation because it detects caspase-3 activity in real-time and also efficiently labels DNA in nuclei of caspase-3-activated cells for real-time fluorescent visualization of apoptotic morphology. The strategy utilized in the design of this fluorogenic substrate can be applied in future endeavors to develop substrates for detecting real-time intracellular enzyme activity.

Enzymatic and chromosomal characterization of HeLa variants.

Seven strains of HeLa cells have been characterized by the number of chromosomes and the activity of the enzymes alkaline phosphatase, glucose-6-phosphate dehydrogenase, 6-phosphogluconic dehydrogenase, and lactic dehydrogenase. All seven strains were found to differ as to chromosome numbers and enzyme levels despite the fact that two strains were called HeLa and three were called HeLa S3. Three strains were found to have a stemline in which greater than 60% of the cells demonstrated a single chromosome number, and this characteristic was stable for at least 6 months. A nomenclature for these clones has been suggested by the use of the stemline chromosome number as a subscript following HeLa. These three clones were, therefore, designated HeLa(65), HeLa(71), and HeLa(75). Karyotypes were made of the stemlines of these clones and were compared with enzyme levels. Alkaline phosphatase showed the greatest variation from cell line to cell line with a 200-fold difference in levels, whereas glucose-6-phosphate dehydrogenase showed variation in activity over a 12-fold range, lactic dehydrogenase over an 8-fold range, and 6-phosphogluconic dehydrogenase over a 2-fold range. It is suggested that human cell strains can be used for biochemical studies if they are cloned and if the clones are relatively stable at least with respect to modal chromosome number and karyotype.

A mitotic form of the Golgi apparatus in HeLa cells.

Galactosyltransferase, a marker for trans-Golgi cisternae in interphase cells, was localized in mitotic HeLa cells embedded in Lowicryl K4M by immunoelectron microscopy. Specific labeling was found only over multivesicular structures that we term Golgi clusters. Unlike Golgi stacks in interphase cells, these clusters lacked elongated cisternae and ordered stacking of their components but did comprise two distinct regions, one containing electron-lucent vesicles and the other, smaller, vesiculo-tubular structures. Labeling for galactosyltransferase was found predominantly over the latter region. Both structures were embedded in a dense matrix that excluded ribosomes and the cluster was often bounded by cisternae of the rough endoplasmic reticulum, sometimes on all sides. Clusters were present at all stages of mitosis examined, which included prometaphase, metaphase, and telophase. They were also identified in conventionally processed mitotic cells and shown to contain another trans-Golgi marker, thiamine pyrophosphatase. Serial sectioning showed that clusters were discrete and globular and multiple copies appeared to be dispersed in the cytoplasm. Their possible role in the division of the Golgi apparatus is discussed.

Immunocytochemical localization of galactosyltransferase in HeLa cells: codistribution with thiamine pyrophosphatase in trans-Golgi cisternae.

An affinity-purified, monospecific rabbit antibody against soluble human milk galactosyltransferase was used to localize the enzyme in HeLa cells by immunofluorescence and by the protein A-gold technique at the electron microscope level. Specific immunofluorescence was observed in a juxtanuclear cytoplasmic region which was identified, on immunostained thin sections of low-temperature Lowicryl K4M-embedded HeLa cells, as Golgi apparatus. Label by gold particles was limited to two to three trans cisternae of the Golgi apparatus, indicating a compartmentalization of galactosyltransferase in the cisternal stack. Combination of preembedding thiamine pyrophosphatase cytochemistry, with postembedding immunostaining for galactosyltransferase proved codistribution of the two enzymes. However, the acid phosphatase-positive, trans-most cisterna was negative for galactosyltransferase. The close topological association of both galactosyltransferase and thiamine pyrophosphatase (or nucleoside diphosphatase) suggests a concerted action of both enzymes in glycosylation.

Cell cycle events in the hydrocortisone regulation of alkaline phosphatase in HeLa S3 cells.

The increase in alkaline phosphatase in asynchronous cultures of HeLa S(3) cells grown in medium supplemented with hydrocortisone is characterized by a lag period of 10-12 hr. Present studies utilizing synchronous cell populations indicate: (a) a minimum of 8-10 hr of incubation with hydrocortisone is necessary for maximum induction of alkaline phosphatase; (b) the increase in enzyme activity produced by hydrocortisone is initiated exclusively in the synthetic phase of the cell cycle; (c) alkaline phosphatase activity does not vary appreciably over a normal control cell cycle. Radioactive hydrocortisone is rapidly distributed into HeLa cells irrespective of their position in the cell cycle, indicating that inductive effects are not governed by selective permeability during the cell cycle. Hydrocortisone-1,2-[(3)H] diffuses back from the cell into the medium when the cells are incubated in fresh medium containing no hydrocortisone, and the alkaline phosphatase induction, under these conditions, is completely reversible.

Amplification of sodium- and potassium-activated adenosinetriphosphatase in HeLa cells by ouabain step selection.

A multistep selection for ouabain resistance was used to isolate a clone of HeLa S3 cells that overproduces the plasma membrane sodium, potassium activated adenosinetriphosphatase (Na+,K+-ATPase). Measurements of specific [3H]ouabain-binding to the resistant clone, C+, and parental HeLa cells indicated that C+ cells contain 8-10 X 10(6) ouabain binding sites per cell compared with 8 X 10(5) per HeLa cell. Plasma membranes isolated from C+ cells by a vesiculation procedure and analyzed for ouabain-dependent incorporation of [32P]phosphate into a 100,000-mol-wt peptide demonstrated a ten- to twelvefold increase in Na+,K+-ATPase catalytic subunit. The affinity of the enzyme for ouabain on the C+ cells was reduced and the time for half maximal ouabain binding was increased compared with the values for the parental cells. The population doubling time for cultures of C+ cells grown in dishes was increased and C+ cells were unable to grow in suspension. Growth of C+ cells in ouabain-free medium resulted in revertant cells, C-, with biochemical and growth properties identical with HeLa. Karyotype analysis revealed that the ouabain-resistant phenotype of the C+ cells was associated with the presence of minute chromosomes which are absent in HeLa and C- cells. This suggests that a gene amplification event is responsible for the Na+,K+-ATPase increase in C+ cells.

Immunocytochemical localization of casein kinase II during interphase and mitosis.

We have developed specific antibodies to synthetic peptide antigens that react with the individual subunits of casein kinase II (CKII). Using these antibodies, we studied the localization of CKII in asynchronous HeLa cells by immunofluorescence and immunoelectron microscopy. Further studies were done on HeLa cells arrested at the G1/S transition by hydroxyurea treatment. Our results indicate that the CKII alpha and beta subunits are localized in the cytoplasm during interphase and are distributed throughout the cell during mitosis. Further electron microscopic investigation revealed that CKII alpha subunit is associated with spindle fibers during metaphase and anaphase. In contrast, the CKII alpha' subunit is localized in the nucleus during G1 and in the cytoplasm during S. Taken together, our results suggest that CKII may play significant roles in cell division control by shifting its localization between the cytoplasm and nucleus.

Protein kinase C mu is located at the Golgi compartment.

Protein kinase C mu (PKC mu) displays unusual structural features like a pleckstrin homology domain and an amino-terminal hydrophobic region with a putative leader peptide and transmembrane sequence. As a discrete location often is a direct clue to the potential biological function of a kinase, antibodies directed against unique amino- and carboxy-terminal domains of PKC mu were used to localize the protein within intracellular compartments in immunofluorescence and subcellular fractionation studies. Confocal laser scanning microscopy showed colocalization of PKC mu with the resident Golgi marker protein beta 1,4 galactosyltransferase in PKC mu transfectants and in the human hepatocellular carcinoma cell line HepG2, expressing endogenous PKC mu. Long-term treatment of cells with brefeldin A, which disintegrates the Golgi apparatus, disrupted PKC mu-specific staining. Cosegregation of PKC mu with beta 1,4 galactosyltransferase, but not with the endosomal marker rab5, upon density gradient fractionation and Western blot analysis of HepG2 cell extracts, provides independent evidence for a Golgi localization of PKC mu. Moreover, cellular sulfate uptake and Golgi-specific glycosaminoglycan sulfation was enhanced in PKC mu transfectants. Together, these data suggest that PKC mu is a resident protein kinase of the core Golgi compartment and is involved in basal transport processes.

Mitochondrial protein synthesis in HeLa cells.

HeLa cell mitochondrial proteins have been shown to be the products of two separate protein-synthesizing systems; one, the general cellular mechanism, sensitive to inhibition by cycloheximide, the other, a specific mitochondrial system subject to inhibition by low concentrations of chloramphenicol (Galper, J. B., and J. E. Darnell. 1971. J. Mol. Biol 57:363). Preliminary data have suggested that a mitochondrial N-formyl-methionyl-tRNA (f-Met-tRNA) might be the initiator tRNA in the latter (Galper, J. B., and J. E. Darnell. 1969. Biochem. Biophys. Res. Commun. 34:205; 1971. J. Mol. Biol. 57:363). It is demonstrated here that the synthesis of these endogenous mitochondrial proteins is also subject to inhibition by ethidium bromide and decays with a half-life of 1(1/2)-2 h in cultures incubated with low concentrations of this dye. The role of formylated f-Met-tRNA as the initiator tRNA in the synthesis of mitochondrial proteins is supported by data from several experiments. The rates of ethidium bromide inhibition of both the charging of f-Met-tRNA and of the synthesis of mitochondrial proteins are strikingly similar. Inhibition by aminopterin of the formylation of f-Met-tRNA greatly depresses the rate of mitochondrial-specific protein synthesis. In the absence of the synthesis of these proteins, respiration, the levels of cytochromes a-a(3) and b, and the number of mitochondrial cristae are decreased. The implications of these findings as they relate to mitochondrial biogenesis are discussed.

Morphological alterations and ganglioside sialyltransferase activity induced by small fatty acids in HeLa cells.

Incubation of HeLa cells in the presence of millimolar concentrations of propionate, butyrate, or pentanoate increases the specific activity of CMP-sialic acid:lactosylceramide sialyltransferase 7-20-fold within 24 h. Longer-chain saturated fatty acids or acetate are much less effective, decanoate showing no induction. Unsaturated fatty acid analogs of butyrate and other compounds are ineffective. Only the three most effective compounds also produce characteristic smooth extended cell processes in HeLa cells. Butyrate (5 mM) induces the sialyltransferase after a 4-h lag, producing maximum specific activity by 24 h. The amount of sialyl-lactosylceramide, the glycolipid product of the enzyme, increases during that time 3.5 times more than in control cultures. No other glycosphingolipid enzyme is significantly altered by butyrate exposure. The cellular shape changes occur 2-3 h later than the increase of sialyltransferase activity, and both processes require the continuous presence of inducer and the synthesis of RNA and protein but not the synthesis of DNA or the presence of serum.