Differentially expressed genes between high-risk human papillomavirus types in human cervical cancer cells.

Cervical carcinoma (CC) is one of the most common cancers among women worldwide and the first cause of death among the Mexican female population. Human papillomavirus (HPV) infection is the most important etiologic factor for CC. Of the oncogenic types, HPV16 and HPV18 are found in 60-70% of invasive CCs worldwide. HPV18 appears to be associated with a more aggressive form of cervical neoplasia than HPV16 infection. At present, there are no studies on differentially expressed cellular genes between transformed cells harboring HPV16 and HPV18 sequences. Based on previous complementary DNA microarray data from our group, 13 genes were found to be differentially overexpressed between HPV16- and HPV18-transformed cells. These genes were as follows: E6BP, UBE4A, C20orf14, ATF7, ABCC8, SLC6A12, WASF3, SUV39H1, SPAG8, CCNC, E2FFE, BIRC5, and DEDD. Differential expression of six selected genes was confirmed by real-time reverse transcription-polymerase chain reaction (RT-PCR). All real-time RT-PCRs confirmed differential expression between HPV18 and HPV(-) samples. The present work identifies genes from signaling pathways triggered by HPV transformation that could be differentially deregulated between HPV16(+) and HPV18(+) samples.

Complexes of Pd(II) and Pt(II) with 9-aminoacridine: reactions with DNA and study of their antiproliferative activity.

Four new metal complexes {M = Pd(II) or Pt(II)} containing the ligand 9-aminoacridine (9AA) were prepared. The compounds were characterized by FT-IR and (1)H, (13)C, and (195)Pt NMR spectroscopies. Crystal structure of the palladium complex of formulae [Pd(9AA)(mu-Cl)](2) . 2DMF was determined by X-ray diffraction. Two 9-acridine molecules in the imine form bind symmetrically to the metal ions in a bidentate fashion through the imine nitrogen atom and the C(1) atom of the aminoacridine closing a new five-membered ring. By reaction with phosphine or pyridine, the Cl bridges broke and compounds with general formulae [Pd(9AA)Cl(L)] (where L = PPh(3) or py) were formed. A mononuclear complex of platinum of formulae [Pt(9AA)Cl(DMSO)] was also obtained by direct reaction of 9-aminoacridine and the complex [PtCl(2)(DMSO(2)]. The capacity of the compounds to modify the secondary and tertiary structures of DNA was evaluated by means of circular dichroism and electrophoretic mobility. Both palladium and platinum compounds proved active in the modification of both the secondary and tertiary DNA structures. AFM images showed noticeable modifications of the morphology of the plasmid pBR322 DNA by the compounds probably due to the intercalation of the complexes between base pairs of the DNA molecule. Finally, the palladium complex was tested for antiproliferative activity against three different human tumor cell lines. The results suggest that the palladium complex of formula [Pd(9AA)(mu-Cl)](2) has significant antiproliferative activity, although it is less active than cisplatin.

Expression profiles of a human pancreatic cancer cell line upon induction of apoptosis search for modulators in cancer therapy.

We analyzed the differential gene expression in the pancreatic cancer cell line NP-18 upon induction of apoptosis caused by cyclin-dependent kinase inhibition triggered by either overexpression of the tumor suppressor gene p16(INK4A)using an adenoviral construction or incubation with the chemical inhibitors, roscovitine or olomoucine. Screening was performed using cDNA arrays from Clontech that allowed the determination of the expression of 1,176 genes specifically related with cancer. The analysis was carried out using the Atlas Image 2.01 (Clontech) and GeneSpring 4.2 (Silicon Genetics) softwares. Among the differentially expressed genes, we chose for further validation histone deacetylase 1 (HDAC1), von Hippel Lindau and decorin as upregulated genes, and Sp1, hypoxia-inducible factor-1 alpha and DNA primase as downregulated genes. The changes in the expression of these genes to mRNA were validated by quantitative RT-PCR and the final translation into protein by Western blot analysis. Inhibition of HDAC activity, Sp1 binding and DNA primase expression led to an increase in the level of apoptosis, both in parental cells and in doxorubicin-resistant cells. Therefore, these proteins could constitute possible targets to develop modulators in cancer chemotherapy that would increase or restore apoptosis.

A novel muscle DNA-binding activity in the GLUT1 promoter.

GLUT1 glucose transporters are highly expressed in proliferating and transformed cells and serum and cAMP or the transcription factor Sp1 induce GLUT1 gene transcription. Here we identified a cis element situated at -46/-37 (MG1E - muscle-specific GLUT1 element) to which muscle-specific nuclear factors bind, and the DNA-protein complexes showed electrophoretic mobility of 41 and 32 kDa. MyoD over-expression induced the generation of MG1E-protein complexes characteristic of myoblast cells. MG1E does not bind any known factors defined in databases. Mutation of the MG1E sequence impaired transcriptional activity of the GLUT1 promoter specifically in skeletal or cardiac muscle cells. The transcriptional activity of the GLUT1 promoter induced by either Sp1, cAMP or serum was markedly reduced when MG1E was inactivated. We propose that the MG1E sequence permits the binding of muscle-specific nuclear factors and a maximal transcriptional activity in muscle cells in response to Sp1, cAMP or serum.

Differential induction of stearoyl-CoA desaturase and acyl-CoA oxidase genes by fibrates in HepG2 cells.

We studied whether two typical effects of fibrates, induction of stearoyl-CoA desaturase (EC 1.14.99.5) and peroxisome proliferation, are related. The effect of bezafibrate on the activity and mRNA of stearoyl-CoA desaturase and acyl-CoA oxidase in the liver and epididymal white adipose tissue of male Sprague-Dawley rats was determined. The same parameters were measured in HepG2 cells, a cell line resistant to peroxisome proliferation, following incubation with ciprofibrate. Bezafibrate increased the hepatic mRNA levels (14.5-fold on day 7) and activity (9.3-fold on day 15) of acyl-CoA oxidase. Stearoyl-CoA desaturase mRNA levels were transiently increased (2.7-fold on day 7), while its activity remained increased at the end of the treatment (2.4-fold). In white adipose tissue, bezafibrate increased the mRNA (5-fold) and activity (1.9-fold) of acyl-CoA oxidase, while stearoyl-CoA desaturase was not modified. Ciprofibrate addition to HepG2 cells cultured in 7% fetal bovine serum (FBS) only increased the stearoyl-CoA desaturase mRNA (1.9-fold). When cells were cultured in 0.5% FBS, ciprofibrate increased acyl-CoA oxidase mRNA (2.2-fold), while the increase in stearoyl-CoA desaturase mRNA was identical (1.9-fold). Further, its activity was also increased (1.5-fold). Incubation of HepG2 cells in the presence of cycloheximide did not alter the capacity of ciprofibrate to induce stearoyl-CoA desaturase mRNA, whereas the presence of actinomycin abolished the induction. In addition, preincubation of HepG2 cells with ciprofibrate increased the rate of stearoyl-CoA desaturase mRNA degradation. The results presented in this study suggest that fibrates induce stearoyl-CoA desaturase activity and mRNA levels independently of peroxisome proliferation.

Differences in the formation of PPARalpha-RXR/acoPPRE complexes between responsive and nonresponsive species upon fibrate administration.

Peroxisome proliferator-activated receptor-alpha (PPARalpha) is responsible for the hypolipidemic, peroxisome proliferation and carcinogenic effects of fibrates. Rats and mice are responsive, but guinea pigs and primates are resistant to the proliferative and carcinogenic effects of these drugs, but the hypolipidemic effect is still manifest. It is not yet clear whether humans should be considered unresponsive, and there is concern about the long-term safety of fibrates. We present molecular evidence for the reported resistance of human cells to peroxisome proliferation by describing a deficient interaction of nuclear extracts from human cells with an acyl-CoA oxidase (ACO)-peroxisome proliferator response element probe upon fibrate addition. Electrophoretic mobility shift assay analysis showed that ciprofibrate elicited a concentration-dependent increase in the binding of nuclear extracts from cells of rat (Morris) and human (HepG2) origin to an ACO-peroxisome proliferator response element probe, although in HepG2 cells the increase was of marginal statistical significance. In Morris cells, the increase was more marked than in HepG2 cells (4-fold versus 1.5-fold at 0.2 mM ciprofibrate), and maximal binding was achieved earlier in Morris (30 min) than in HepG2 cells (3 h). Morris cells responded to the addition of ciprofibrate by increasing the levels of ACO mRNA, whereas HepG2 did not. The ratio between PPARbeta/PPARalpha mRNAs was higher in HepG2 cells than in Morris cells (3.2 versus 1.9), pointing to an antagonizing effect of PPARbeta on PPARalpha activity. These results were obtained in untransfected cells expressing their own basal set of receptors. We also provide evidence of the translocation of PPARalpha from the cytosol to the nucleus upon activation by ciprofibrate.

Identification by RNA-based arbitrarily primed PCR of the involvement of cytochrome c oxidase in the development of resistance to methotrexate.

RNA-based arbitrarily primed PCR (RAP-PCR) was used to identify sequences in CHO K1 cells that were differentially expressed upon methotrexate incubation during the development of resistance to this drug. Ten different RAP products were isolated, cloned and sequenced. Among these, we identified one sequence that showed 84% identity with the nucleotide sequence of rat cytochrome c oxidase subunit II, and 90% identity with the amino acid sequence of this protein. This RAP fragment was up-regulated in a dose- and time-dependent manner. The overexpression of cytochrome c oxidase subunit II mRNA as a result of methotrexate incubation was corroborated by quantitative RT-PCR and Northern blot analysis. Incubation of cells with sodium azide, a specific cytochrome c oxidase inhibitor, decreased the number of resistant colonies after methotrexate treatment. Thus, overexpression of cytochrome c oxidase is involved in the development of resistance to methotrexate. These results suggest that sodium azide may be used as a modulator in chemotherapy with methotrexate.

Effects of anti-sense oligonucleotides directed toward dihydrofolate reductase RNA in mammalian cultured cells.

The effect of incubations with anti-sense phosphorothioate oligonucleotides directed toward sequences of dihydrofolate reductase (DHFR) RNA has been tested on Chinese hamster ovary cells. The selected targets were the 5'-untranslated region, the translational start, the splice sites and branch point of intron I and polyadenylation regions 1 and 3 of the DHFR RNA. To introduce the oligonucleotides, the cationic liposome DOTAP was used. The oligonucleotides most effective at causing cytotoxicity were ATNL and DTNL, both directed toward the translation-start site, at a range of concentrations between 1 and 4 microM. The minimum time for the oligonucleotide to exert its full cytotoxic effect was 3 days. Excess of oligonucleotide diminished the cytotoxic effect. Oligonucleotide uptake was monitored by the incorporation of [32P]- or fluorescein-labeled oligonucleotide and was found to depend on liposome and oligonucleotide concentrations and duration of incubation. Formation of in vitro complexes between the oligonucleotide and the liposome was also studied. Cytotoxicity was observed when the oligonucleotide was incubated with cell lines containing either the endogenous gene or co-transfected DHFR minigenes. Cell incubation with ATNL caused a time-dependent decrease in the levels of DHFR mRNA and enzymatic activity. Moreover, a cell line bearing amplification at the dhfr locus was equally affected by the action of ATNL. Human hepatoma cells were also affected by treatment with the counterpart of ATNL in the human DHFR mRNA sequence. Our results set the basis for a possible cancer therapy with anti-sense oligonucleotides using DHFR as the target.

Retinoblastoma protein associates with SP1 and activates the hamster dihydrofolate reductase promoter.

The dihydrofolate reductase (dhfr) promoter is powerfully activated by the transcription factor Sp1. It has been suggested that Sp1 is a potential target for transcriptional regulation by the cell cycle regulator retinoblastoma protein (Rb), and so we have explored this possibility using the hamster dhfr gene as a model. By the use of DNA probes from the hamster dhfr gene promoter, containing the most proximal GC box (minimal promoter), and nuclear extracts from cultured hamster cells (CHO K1), we show that polyclonal and monoclonal antibodies against Rb supershift the binding of Sp1. Nuclear extract immunoprecipitation with anti-Rb followed by Western analysis using anti-Sp1 also shows that Rb is complexed to Sp1. Complementary Immunoprecipitation/WB analysis shows both forms of Rb protein in the anti-Sp1 immunoprecipitates. Moreover, nuclear extract immunodepletion of Rb abolishes Sp1 gel-shift. The interaction between Rb and Sp1 is maintained in all the phases of the cell cycle. Transient overexpression of Rb in dhfr negative cells co-transfected with a dhfr minigene driven by its minimal promoter increases DHFR activity and potentiates transcription when overexpressing Sp1. Both effects are severely reduced when the co-transfections are performed with a homologous dhfr minigene containing a single point mutation in the GC box. Thus, the activation by Rb of the dhfr gene may be exerted through Sp1. Stable transfectants of pCMVRb in K1 cells show an increase in both mRNA and DHFR activity. It is concluded that Sp1 is physically associated with Rb, and that this association increases Sp1-mediated transcription of the hamster dhfr gene.

Cell-growth regulation of the hamster dihydrofolate reductase gene promoter by transcription factor Sp1.

The dihydrofolate reductase (DHFR) gene (dhfr) promoter contains cis-acting elements for the transcription factors Sp1 and E2F. Given the ability of Sp1 to activate the dhfr promoter, we have evaluated the contribution of Sp1 to the cell-growth regulation of the dhfr gene. Using gel-mobility assays performed with DNA probes from the minimal promoter of the hamster dhfr gene and nuclear extracts from cultured hamster cells (CHO K1) we show that the binding of Sp1 to the dhfr promoter is cell-growth-phase regulated. Accordingly, dhfr transcription and mRNA levels in K1 cells increase upon serum stimulation. Cytological detection of Sp1 by immunofluorescence reveals a decrease of this protein in the process leading to the G0 state, and an increase upon serum stimulation of quiescent cells. These results were confirmed by western blot analysis. It is concluded that Sp1 progressively binds to the hamster dhfr promoter after stimulation of cell proliferation, which can account for the transcriptional regulation of the dhfr gene during the cell cycle. The role of Sp1 in the specific control of dhfr during the cell cycle was confirmed in vivo using cell lines derived from dhfr-negative cells transfected with dhfr plasmids carrying either the wild-type or mutated Sp1-binding or E2F-binding sites in the dhfr minimal promoter.

Purine enzyme profile in human colon-carcinoma cell lines and differential sensitivity to deoxycoformycin and 2'-deoxyadenosine in combination.

Different cell lines, 2 from human colon carcinoma (LoVo and HT29) and 1 from Chinese hamster ovary (CHO K-I), were examined to assess the effect of deoxycoformycin (dCF), an inhibitor of adenosine deaminase (ADA), and 2'-deoxyadenosine (dAdo) on their growth. When used alone, neither dCF or dAdo were cytotoxic for the 3 cell lines, while their combination caused inhibition of cell growth, with the following sensitivity: CHO K-I > LoVo > HT29. We studied the pattern of enzymatic activities involved in the metabolism of dAdo in the 3 cell lines. The phosphorylation of dAdo by adenosine kinase appears to play a central role in the toxicity of the deoxynucleoside in combination with dCF. In fact, CHO K-I cells, which are the most sensitive, possess the highest level of this enzyme. Moreover, the cytotoxic effect was almost completely reversed in the 3 cell lines when inhibitors of adenosine kinase, such as 5'-amino-5'-deoxyadenosine and iodotubercidine, were added to the culture medium together with dCF and dAdo. In addition, baby hamster kidney (BHK) adenosine-kinase-deficient (AK-) cells were highly resistant to this treatment. Uptake inhibition of dAdo using dipyridamole also caused reversal of the toxicity. The AMP and deoxyAMP dephosphorylating activities, much lower in the CHO K-I cells, also appear to play a central role in the toxicity of dAdo when adenosine deaminase is inhibited. However, our data suggest that other factors may modulate the toxic effect, such as S-adenosyl-homocysteine-hydrolase inhibition by dAdo at high concentrations.

Regulation of mitochondrial 3-hydroxy-3-methylglutaryl-coenzyme A synthase protein by starvation, fat feeding, and diabetes.

We have determined the levels of mitochondrial 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase under different metabolic situations to examine its potential role as a regulatory protein in the ketogenic pathway. We used specific antibodies directed against a peptide of the amino acid sequence of the protein as deduced from the cDNA sequence. The amount of mitochondrial HMG-CoA synthase protein rapidly increased in response to cyclic AMP, dexamethasone, starvation, fat feeding, and diabetes, whereas it was decreased by insulin and refeeding. Insulin was also able to counteract the increase in mitochondrial HMG-CoA synthase levels observed under the diabetic condition. Furthermore, the finding that quantitative changes in HMG-CoA synthase protein were less marked than those in the corresponding mRNA in starved and diabetic rats suggests either translational control or increased degradation of either mRNA or protein. All these results indicate that mitochondrial HMG-CoA synthase is a regulatory element in the ketogenic process.

Role of AMP on the activation of glycogen synthase and phosphorylase by adenosine, fructose, and glutamine in rat hepatocytes.

The mechanism for glycogen synthesis stimulation produced by adenosine, fructose, and glutamine has been investigated. We have analyzed the relationship between adenine nucleotides and glycogen metabolism rate-limiting enzymes upon hepatocyte incubation with these three compounds. In isolated hepatocytes, inhibition of AMP deaminase with erythro-9-(2-hydroxyl-3nonyl)adenine further increases the accumulation of AMP and the activation of glycogen synthase and phosphorylase by fructose. This ketose does not increase cyclic AMP or the activity of cyclic AMP-dependent protein kinase. Adenosine raises AMP and ATP concentration. This nucleotide also activates glycogen synthase and phosphorylase by covalent modification. The correlation coefficient between AMP and glycogen synthase activity is 0.974. Nitrobenzylthioinosine, a transport inhibitor of adenosine, blocks (by 50%) the effect of the nucleoside on AMP formation and glycogen synthase but not on phosphorylase. 2-Chloroadenosine and N6-phenylisopropyladenosine, nonmetabolizable analogues of adenosine, activate phosphorylase (6-fold) without increasing the concentration of adenine nucleotides or the activity of glycogen synthase. Cyclic AMP is not increased by adenosine in hepatocytes from starved rats but is in cells from fed animals. [Ethylenebis (oxyethylenenitrilo)]tetraacetic acid (EGTA) blocks by 60% the activation of phosphorylase by adenosine but not that of glycogen synthase. Glutamine also increases AMP concentration and glycogen synthase and phosphorylase activities, and these effects are blocked by 6-mercaptopurine, a purine synthesis inhibitor. Neither adenosine nor glutamine increases glucose 6-phosphate. It is proposed that the observed efficient glycogen synthesis from fructose, adenosine, and glutamine is due to the generation of AMP that activates glycogen synthase probably through increases in synthase phosphatase activity. It is also concluded that the activation of phosphorylase by the above-mentioned compounds can be triggered by metabolic changes.

Glycogen synthesis from glucose and fructose in hepatocytes from diabetic rats.

In rat hepatocytes, the basal glycogen synthase activation state is decreased in the fed and diabetic states, whereas glycogen phosphorylase a activity decreases only in diabetes. Diabetes practically abolishes the time- and dose-dependent activation of glycogen synthase to glucose especially in the fed state. Fructose, however, is still able to activate this enzyme. Glycogen phosphorylase response to both sugars is operative in all cases. Cell incubation with the combination of 20 mM glucose plus 3 mM fructose produces a great activation of glycogen synthase and a potentiated glycogen deposition in both normal and diabetic conditions. Using radiolabeled sugars, we demonstrate that this enhanced glycogen synthesis is achieved from both glucose and fructose even in the diabetic state. Therefore, the presence of fructose plays a permissive role in glycogen synthesis from glucose in diabetic animals. Glucose and fructose increase the intracellular concentration of glucose 6-phosphate and fructose reduces the concentration of ATP. There is a close correlation between the ratio of the intracellular concentrations of glucose 6-phosphate and ATP (G6-P/ATP) and the activation state of glycogen synthase in hepatocytes from both normal and diabetic animals. However, for any given value of the G6-P/ATP ratio, the activation state of glycogen synthase in diabetic animals is always lower than that of normal animals. This suggests that the system that activates glycogen synthase (synthase phosphatase activity) is impaired in the diabetic state. The permissive effect of fructose is probably exerted through its capacity to increase the G6-P/ATP ratio which may partially increase synthase phosphatase activity, rendering glycogen synthase active.

Glycogen synthase activation by sugars in isolated hepatocytes.

We have investigated the activation by sugars of glycogen synthase in relation to (i) phosphorylase a activity and (ii) changes in the intracellular concentration of glucose 6-phosphate and adenine nucleotides. All the sugars tested in this work present the common denominator of activating glycogen synthase. On the other hand, phosphorylase a activity is decreased by mannose and glucose, unchanged by galactose and xylitol, and increased by tagatose, glyceraldehyde, and fructose. Dihydroxyacetone exerts a biphasic effect on phosphorylase. These findings provide additional evidence proving that glycogen synthase can be activated regardless of the levels of phosphorylase a, clearly establishing that a nonsequential mechanism for the activation of glycogen synthase occurs in liver cells. The glycogen synthase activation state is related to the concentrations of glucose 6-phosphate and adenine nucleotides. In this respect, tagatose, glyceraldehyde, and fructose deplete ATP and increase AMP contents, whereas glucose, mannose, galactose, xylitol, and dihydroxyacetone do not alter the concentration of these nucleotides. In addition, all these sugars, except glyceraldehyde, increase the intracellular content of glucose 6-phosphate. The activation of glycogen synthase by sugars is reflected in decreases on both kinetic constants of the enzyme, M0.5 (for glucose 6-phosphate) and S0.5 (for UDP-glucose). We propose that hepatocyte glycogen synthase is activated by monosaccharides by a mechanism triggered by changes in glucose 6-phosphate and adenine nucleotide concentrations which have been described to modify glycogen synthase phosphatase activity. This mechanism represents a metabolite control of the sugar-induced activation of hepatocyte glycogen synthase.

Effects of glucagon and insulin on the cyclic AMP binding capacity of hepatocyte cyclic AMP-dependent protein kinase.

Extracts obtained from rat hepatocytes incubated with saline, glucagon or insulin were electrophoresed on polyacrylamide gels and then assayed for cyclic (3H)AMP binding capacity. Analysis of the binding patterns demonstrated that glucagon dissociated a holoenzyme of cyclic AMP-dependent protein kinase in a dose-dependent manner. The increase in free regulatory subunits and, hence, in free catalytic subunits explains the activation of this enzyme by glucagon in the liver. Insulin decreased both the amount of cyclic (3H)AMP bound to the holoenzyme and the capacity of the enzyme to be dissociated when the extracts were incubated with increasing concentrations of this cyclic nucleotide. We propose that these insulin-induced effects are determined by an inhibition of the cyclic AMP binding capacity of this protein kinase. This mechanism could account for the inactivation of cyclic AMP-dependent protein kinase that insulin causes in the liver.

Glucose 6-phosphate plays a central role in the activation of glycogen synthase by glucose in hepatocytes.

The state of activation of glycogen synthase enhanced by glucose, other sugars and gluconeogenic precursors shows a strong positive correlation with the intracellular concentrations of glucose 6-P when ATP concentrations remain constant. The concentrations of glucose 6-P achieved upon incubation of hepatocytes with glucose plus mannoheptulose, an inhibitor of glucokinase and hexokinase, were lower than those found when the incubation was carried out with glucose alone. Under these conditions, in keeping with the decrease in glucose 6-P, the activation of glycogen synthase by glucose was also impaired. On the other hand the inactivation of glycogen phosphorylase was not altered in the presence of mannoheptulose.

Activation of hepatocyte glycogen synthase by metabolic inhibitors.

Incubation of isolated rat hepatocytes with metabolic inhibitors causes an increase in the -glucose 6-P/+glucose 6-P activity ratio of glycogen synthase after decreasing ATP and increasing AMP levels. Concomitantly, the activity of phosphorylase is increased six-fold by the same treatment. This activation of both enzymes remains after gel filtration of the hepatocyte extracts. Addition of metabolic inhibitors to cells pretreated with an inhibitor of AMP-deaminase results in an accumulation of AMP and, simultaneously, in a further increase in the activation state of glycogen synthase. The correlation coefficient between the intracellular concentration of AMP and glycogen synthase activity is r = 0.93. It is proposed that the covalent activation of glycogen synthase by metabolic inhibitors can be triggered by changes in the level of the intracellular concentrations of adenine nucleotides.