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.

Sp1 involvement in the 4beta-phorbol 12-myristate 13-acetate (TPA)-mediated increase in resistance to methotrexate in Chinese hamster ovary cells.

4beta-Phorbol 12-myristate 13-acetate (TPA) increases the number of colonies resistant to methotrexate (MTX), mainly by amplification of the dihydrofolate reductase (dhfr) locus. We showed previously that inhibition of protein kinase C (PKC) prevents this resistance. Here, we studied the molecular changes involved in the development of TPA-mediated MTX resistance in Chinese hamster ovary (CHO) cells. TPA incubation increased the expression and activity of DHFR. Because Sp1 controls the dhfr promoter, we determined the effect of TPA on the expression of Sp1 and its binding to DNA. TPA incubation increased Sp1 binding and the levels of Sp1 protein. The latter effect was due to an increase in Sp1 mRNA. Dephosphorylation of nuclear extracts from control or TPA-treated cells reduced the binding of Sp1. Stable transfectants of PKCalpha showed increased Sp1 binding, and when treated with MTX, developed a greater number of resistant colonies than control cells. Seventy-five percent of the isolated colonies showed increased copy number for the dhfr gene. Transient expression of PKCalpha increased DHFR activity. Over-expression of Sp1 increased resistance to MTX, and inhibition of Sp1 binding by mithramycin decreased this resistance. We conclude that one mechanism by which TPA enhances MTX resistance, mainly by gene amplification, is through an increase in Sp1 expression which leads to DHFR activation.

Cloning and characterization of the 5'-flanking region of the human transcription factor Sp1 gene.

The 5'-flanking region of the human Sp1 gene was cloned and characterized. Sequence analysis of this region showed the absence of both CAAT and TATA boxes and an initiator element. The proximal promoter of the Sp1 gene is a GC-rich region that contains multiple GC boxes and Ap2 binding sites. The major transcription start site is located 63 base pairs upstream of the translation start site. Transfection experiments demonstrate that all the elements necessary to achieve significant basal transcription activity are located between positions -443 and -20 relative to the translational start. Sp1 and Sp3 proteins bind to the downstream GC box located in the proximal promoter of Sp1. Furthermore, we demonstrate that the Sp1 protein activates Sp1 transcription activity; thus the Sp1 gene is autoregulated.

DAG accumulation from saturated fatty acids desensitizes insulin stimulation of glucose uptake in muscle cells.

The increased availability of saturated lipids has been correlated with development of insulin resistance, although the basis for this impairment is not defined. This work examined the interaction of saturated and unsaturated fatty acids (FA) with insulin stimulation of glucose uptake and its relation to the FA incorporation into different lipid pools in cultured human muscle. It is shown that basal or insulin-stimulated 2-deoxyglucose uptake was unaltered in cells preincubated with oleate, whereas basal glucose uptake was increased and insulin response was impaired in palmitate- and stearate-loaded cells. Analysis of the incorporation of FA into different lipid pools showed that palmitate, stearate, and oleate were similarly incorporated into phospholipids (PL) and did not modify the FA profile. In contrast, differences were observed in the total incorporation of FA into triacylglycerides (TAG): unsaturated FA were readily diverted toward TAG, whereas saturated FA could accumulate as diacylglycerol (DAG). Treatment with palmitate increased the activity of membrane-associated protein kinase C, whereas oleate had no effect. Mixture of palmitate with oleate diverted the saturated FA toward TAG and abolished its effect on glucose uptake. In conclusion, our data indicate that saturated FA-promoted changes in basal glucose uptake and insulin response were not correlated to a modification of the FA profile in PL or TAG accumulation. In contrast, these changes were related to saturated FA being accumulated as DAG and activating protein kinase C. Therefore, our results suggest that accumulation of DAG may be a molecular link between an increased availability of saturated FA and the induction of insulin resistance.

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.

Effect of differential polyadenylation and cell growth phase on dihydrofolate reductase mRNA stability.

We have constructed tetracycline-responsive dhfr minigenes and transferred them to a Chinese hamster ovary cell DHFR-deficient deletion mutant to obtained cells in which dhfr transcription can be repressed by tetracycline (tet-off). DHFR mRNA half-life measured after the repression of transcription by tetracycline in these transfectants is about 1.5 h, which is significantly shorter than previously reported. In addition, we observed that DHFR mRNA is less stable in serum-starved cells than in exponentially growing cells. Given that the dhfr gene contains multiple polyadenylation sites, we analyzed the role of polyadenylation site usage on the stability of the resultant mRNA molecules. We found that DHFR mRNA is more stable when a strong polyadenylation site is used. Finally, we have observed that the relative lengths of the poly(A) tails for the different DHFR mRNA species correlated with their relative stability in growing versus resting cells.

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.

Protein kinase C inhibitors reduce phorbol ester-induced resistance to methotrexate in Chinese hamster ovary cells.

Phorbol 12-myristate 13-acetate (TPA) increases the number of colonies surviving methotrexate (MTX) exposure in a dose-dependent manner upon short incubation with Chinese hamster ovary (CHO) cells. Seventy percent of the isolated colonies showed increased copy number for the dihydrofolate reductase gene. EGTA prevents the increase in resistance triggered by TPA. Calcium ionophore A23187 and angiotensin II also increase this resistance, suggesting that calcium is involved in this process. Protein kinase C (PKC) from CHO cells is rapidly activated by TPA, A23187 and angiotensin II. PKC inhibitors, 1-(5-Isoquinolinylsulphonyl)-2-methyl-piperazine (H-7), glycyrrhetinic acid, staurosporine and calphostin C decrease the generation of resistant colonies to MTX upon incubation with TPA. However, 5 nM staurosporine on its own increases resistance to MTX while having the ability to translocate CHO PKC. In vitro, H-7, staurosporine and calphostin C inhibit PKC activity translocated by TPA incubation with CHO cells. We conclude that PKC, the activity of which is dependent on calcium and phospholipids, is part of the pathway that leads to development of increased resistance to MTX. Thus, inhibition of PKC prevents the appearance of this resistance. Our results suggest the possibility of using non-toxic PKC inhibitors as resistance modulators in MTX chemotherapy.

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.

Regulation of the expression of the mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase gene. Its role in the control of ketogenesis.

We have explored the role of mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase in regulating ketogenesis. We had previously cloned the cDNA for mitochondrial HMG-CoA synthase and have now studied the regulation in vivo of the expression of this gene in rat liver. The amount of processed mitochondrial HMG-CoA synthase mRNA is rapidly changed in response to cyclic AMP, insulin, dexamethasone and refeeding, and is greatly increased by starvation, fat feeding and diabetes. We conclude that one point of ketogenic control is exercised at the level of genetic expression of mitochondrial HMG-CoA synthase.