Nuclear domain 10-associated proteins recognize and segregate intranuclear DNA/protein complexes to negate gene expression.

BACKGROUND: DNA viruses, such as herpes simplex virus type 1 (HSV-1), Simian virus 40 (SV40), and Cytomegaloviruses (CMV), start their replicative processes and transcription at specific nuclear domains known as ND10 (nuclear domain 10, also called PML bodies). It has been previously determined that for HSV-1 and SV40, a short DNA sequence and its binding protein are required and sufficient for cell localization of viral DNA replication and gene transcription. RESULTS: Our recent observations provide evidence that a foreign (not endogenous) DNA/protein complex in the nucleus recruits ND10 proteins. First, the complexes formed from the bacterial lac operator DNA and its binding protein (lac repressor), or from HPV11 (human papillomavirus 11) origin DNA and its binding protein (E2), co-localized with different ND10 proteins. Second, the HSV-1 amplicon without inserted lac operator DNA repeats distributed in the nucleus randomly, whereas the amplicon with lac operator DNA repeats associated with ND10, suggesting that DNA-binding proteins are required to localize at ND10. The cellular intrinsic DNA/protein complex (as detected for U2 DNA) showed no association with ND10. Furthermore, our examination of PML-/-, Daxx-/-, and Sp100-negative cells led to our discovering that DNA/protein complexes recruit ND10 protein independently. Using the GFP-LacI/Operator system, we were able to direct the transfected DNA to ND10 and found that gene expression was significantly repressed when the transfected DNA was directed to ND10. CONCLUSION: Taken together, the results suggest that cells recognize DNA/protein complexes through a mechanism that involves interaction with the ND10-associated proteins.

Assessment of infection with polyomaviruses BKV, JCV and SV40 in different groups of Cuban individuals.

We investigated the frequency of BKV, JCV and SV40 reactivation in three groups of Cuban patients by multiplex nested PCR assay of 40 paraffin-embedded colorectal neoplasm tissues, 113 urine samples, and 125 plasma samples from 27 transplant recipients, and cerebrospinal fluid (CSF) from 67 HIV-1-infected individuals with central nervous system (CNS) disorders. None of these polyomaviruses were detected in colorectal neoplasms. JCV DNA was detected in 2 of 67 patients (2.9%) with CNS disorders, but neither BKV nor SV40 was identified. BKV was found in urine from 38.5% and 28.6% of adult and pediatric transplant recipients, respectively. In adult renal transplant recipients, excretion of BKV in urine was significantly associated with episodes of acute rejection (p=0.012) and with excretion of HCMV in urine (p= 0.008). In Cuba, the polyomaviruses studied here could not be related to colorectal neoplasms, and JCV was rarely detected in CSFs of HIV-1-infected individuals, whilst BKV reactivation was found to occur frequently in organ transplant recipients.

Effect of mutations in a simian virus 40 PolyA signal enhancer on green fluorescent protein reporter gene expression.

Our previous studies have shown that tandem Alu repeats inhibit green fluorescent protein (GFP) gene expression when inserted downstream of the GFP gene in the pEGFP-C1 vector. We found that the 22R sequence (5'-GTGAAAAAAATGCTTTATTTGT-3') from the antisense PolyA (240 bp polyadenylation signal) of simian virus 40, eliminated repression of GFP gene expression when inserted between the GFP gene and the Alu repeats. The 22R sequence contains an imperfect palindrome; based on RNA structure software prediction, it forms an unstable stem-loop structure, including a loop, a first stem, a bulge, and a second stem. Analysis of mutations of the loop length of the 22R sequence showed that the three-nucleotide loop (wild-type, 22R) induced much stronger GFP expression than did other loop lengths. Two mutations, 4TMI (A7→T, A17→T) and 5AMI (A6→T, T18→A), which caused the base type changes in the bulge and in the second stem in the 22R sequence, induced stronger GFP gene expression than 22R itself. Mutation of the bulge base (A17→T), leading to complete complementation of the stem, caused weaker GFP gene expression. Sequences without a palindrome (7pieA, 5'-GTGAAAAAAATG CAAAAAAAGT-3', 7pieT, 5'-GTGTTTTTTTTGCTTTTTTTGT-3') did not activate GFP gene expression. We conclude that an imperfect palindrome affects and can increase GFP gene expression.

Gene expression promoted by the SV40 DNA targeting sequence and the hypoxia-responsive element under normoxia and hypoxia.

The main objective of the present study was to find suitable DNA-targeting sequences (DTS) for the construction of plasmid vectors to be used to treat ischemic diseases. The well-known Simian virus 40 nuclear DTS (SV40-DTS) and hypoxia-responsive element (HRE) sequences were used to construct plasmid vectors to express the human vascular endothelial growth factor gene (hVEGF). The rate of plasmid nuclear transport and consequent gene expression under normoxia (20% O2) and hypoxia (less than 5% O2) were determined. Plasmids containing the SV40-DTS or HRE sequences were constructed and used to transfect the A293T cell line (a human embryonic kidney cell line) in vitro and mouse skeletal muscle cells in vivo. Plasmid transport to the nucleus was monitored by real-time PCR, and the expression level of the hVEGF gene was measured by ELISA. The in vitro nuclear transport efficiency of the SV40-DTS plasmid was about 50% lower under hypoxia, while the HRE plasmid was about 50% higher under hypoxia. Quantitation of reporter gene expression in vitro and in vivo, under hypoxia and normoxia, confirmed that the SV40-DTS plasmid functioned better under normoxia, while the HRE plasmid was superior under hypoxia. These results indicate that the efficiency of gene expression by plasmids containing DNA binding sequences is affected by the concentration of oxygen in the medium.

Identification of viral infections in the prostate and evaluation of their association with cancer.

BACKGROUND: Several viruses with known oncogenic potential infect prostate tissue, among these are the polyomaviruses BKV, JCV, and SV40; human papillomaviruses (HPVs), and human cytomegalovirus (HCMV) infections. Recently, the Xenotropic Murine Leukemia Virus-related gammaretrovirus (XMRV) was identified in prostate tissue with a high prevalence observed in prostate cancer (PC) patients homozygous for the glutamine variant of the RNASEL protein (462Q/Q). Association studies with the R462Q allele and non-XMRV viruses have not been reported. We assessed associations between prostate cancer, prostate viral infections, and the RNASEL 462Q allele in Mexican cancer patients and controls. METHODS: 130 subjects (55 prostate cancer cases and 75 controls) were enrolled in the study. DNA and RNA isolated from prostate tissues were screened for the presence of viral genomes. Genotyping of the RNASEL R462Q variant was performed by Taqman method. RESULTS: R/R, R/Q, and Q/Q frequencies for R462Q were 0.62, 0.38, and 0.0 for PC cases and 0.69, 0.24, and 0.07 for controls, respectively. HPV sequences were detected in 11 (20.0%) cases and 4 (5.3%) controls. XMRV and HCMV infections were detected in one and six control samples, respectively. The risk of PC was significantly increased (Odds Ratio = 3.98; 95% CI: 1.17-13.56, p = 0.027) by infection of the prostatic tissue with HPV. BKV, JCV, and SV40 sequences were not detected in any of the tissue samples examined. CONCLUSIONS: We report a positive association between PC and HPV infection. The 462Q/Q RNASEL genotype was not represented in our PC cases; thus, its interaction with prostate viral infections and cancer could not be evaluated.

Signaling pathways involved with the stimulatory effect of angiotensin II on vacuolar H+-ATPase in proximal tubule cells.

It has been documented that angiotensin II (ANG II) (10(-9) M) stimulates proton extrusion via H(+)-adenosine triphosphatase (ATPase) in proximal tubule cells. In the present study, we investigated the signaling pathways involved in the effects of ANG II on H(+)-ATPase activity and on the cytosolic free calcium concentration in immortalized rat proximal tubule cells, a permanent cell line derived from rat proximal tubules. The effects of ANG on pH(i) and [Ca(+2)](i) were assessed by the fluorescent probes, 2',7-bis (2-carboxyethyl)-5(6)-carboxyfluorescein-acetoxy-methyl ester and fluo-4-acetoxy-methyl ester, in the absence of Na(+) to block the Na(+)/H(+) exchanger. In the control situation, the pH recovery rate following intracellular acidification with NH(4)Cl was 0.073+/-0.011 pH units/min (n=12). This recovery was significantly increased with ANG II (10(-9 )M), to 0.12+/-0.015 pH units/min, n=10. This last effect was also followed by a significant increase of Ca(+2) (i), from 99.72+/-1.704 nM (n=21) to 401.23+/-33.91 nM (n=39). The stimulatory effect of ANG II was blocked in the presence of losartan, an angiotensin II subtype 1 (AT(1)) receptor antagonist. H89 [protein kinase A (PKA) inhibitor] plus ANG II had no effect on the pH recovery. Staurosporine [protein kinase C (PKC) inhibitor] impaired the effect of ANG II. Phorbol myristate acetate (PKC activator) mimicked in part the stimulatory effect of ANG II, but reduced Ca(+2) (i). 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (intracellular calcium chelator) alone reduced the pH(i) recovery rate below control levels and impaired the effect of ANG II, in a way similar to that of trimethoxy benzoate (a blocker of Ca(+2) (i) mobilization). We conclude that ANG II regulates rat proximal tubule vacuolar H(+)-ATPase by a PKA-independent mechanism and that PKC and intracellular calcium play a critical role in this regulation.

Establishment and partial characterization of an ovine synovial membrane cell line obtained by transformation with Simian Virus 40 T antigen.

The small ruminant lentiviruses, namely caprine arthritis encephalitis virus (CAEV) and Maedi Visna virus (MVV) are grown currently in secondary synovial membrane cells. Primary and secondary cell cultures are sometimes difficult to obtain and support a low number of passages and, therefore, permissive cell lines are needed. A transformed cell line was obtained by transfection of ovine synovial membrane secondary cell culture with a plasmid containing the SV40 large T antigen gene. The transformed cell culture described in this paper showed a higher growth rate and a more homogenous population of fibroblast-like cells when compared to the original ovine synovial membrane secondary cell cultures. Karyotype analysis has indicated the induction of many random chromosome changes, leading to a decrease in chromosome number. The SV40 DNA was detected in the nucleus and in the cytoplasm of transformed cells. The putative expression of large T antigen was presumed by the detection of the corresponding mRNA by PCR. Finally, the transformed ovine synovial membrane cells were shown to be permissive to small ruminant lentiviruses, and these are suggested as a cell line for in vitro isolation and propagation of these viruses.

High stearoyl-CoA desaturase protein and activity levels in simian virus 40 transformed-human lung fibroblasts.

The precise role of monounsaturated fatty acid (MUFA) synthesis in cell proliferation and programmed cell death remains unknown. The strong correlation of high levels of MUFA and neoplastic phenotype suggest that the regulation of stearoyl CoA desaturase (SCD) must play a significant role in cancer development. In this study, the levels of SCD protein and activity were investigated in normal (WI38) and SV40-transformed (SV40-WI38) human lung fibroblasts. Thus, the activity of SCD on exogenous [14C]stearic acid and endogenous [14C]acetate-labeled fatty acids was increased by 2.2- and 2.6-fold, respectively, in SV40-WI38 compared to WI38 fibroblasts. Concomitantly, a 3.3-fold increase in SCD protein content was observed in SV40-transformed cells. Cell transformation also led to high levels of MUFA, which was paralleled by a more fluid membrane environment. Furthermore, the levels of PPAR-gamma, a well-known activator of SCD expression, were highly increased in SV40-transformed fibroblasts. SCD activity appeared linked to the events of programmed cell death, since incubations with 40 microM etoposide induced apoptosis in SV40 cells, and led to a decrease in fatty acid synthesis, SCD activity and in MUFA cellular levels. Taken together, these results suggest that SCD protein and activity levels are associated with the events of neoplastic cell transformation and programmed cell death.

Transcriptional activators differ in their abilities to control alternative splicing.

Promoter and enhancer elements can influence alternative splicing, but the basis for this phenomenon is not well understood. Here we investigated how different transcriptional activators affect the decision between inclusion and exclusion (skipping) of the fibronectin EDI exon. A mutant of the acidic VP16 activation domain called SW6 that preferentially inhibits polymerase II (pol II) elongation caused a reduction in EDI exon skipping. Exon skipping was fully restored in the presence of the SW6 mutant by either the SV40 enhancer in cis or the human immunodeficiency virus (HIV) Tat in trans, both of which specifically stimulate pol II elongation. HIV Tat also cooperated with the Sp1 and CTF activation domains to enhance transcript elongation and EDI skipping. The extent of exon skipping correlated with the efficiency with which pol II transcripts reach the 3' end of the gene but not with the overall fold increase in transcript levels caused by different activators. The ability of activators to enhance elongation by RNA polymerase II therefore correlates with their ability to enhance exon skipping. Consistent with this observation, the elongation inhibitor dichlororibofuranosylbenzimidazole (DRB) enhanced EDI inclusion. Conversely, the histone deacetylase inhibitor trichostatin A that is thought to stimulate elongation caused a modest inhibition of EDI inclusion. Together our results support a kinetic coupling model in which the rate of transcript elongation determines the outcome of two competing splicing reactions that occur co-transcriptionally. Rapid, highly processive transcription favors EDI exon skipping, whereas slower, less processive transcription favors inclusion.

Regulation of alternative splicing by a transcriptional enhancer through RNA pol II elongation.

Promoters and enhancers are cis-acting elements that control gene transcription via complex networks of protein-DNA and protein-protein interactions. Whereas promoters deal with putting in place the RNA polymerase, both enhancers and promoters can control transcriptional initiation and elongation. We have previously shown that promoter structure modulates alternative splicing, strengthening the concept of a physical and functional coupling between transcription and splicing. Here we report that the promoter effect is due to the control of RNA pol II elongation. We found that the simian virus 40 (SV40) transcriptional enhancer, inserted in fibronectin (FN) minigene constructs transfected into mammalian cells, controls alternative splicing by inhibiting inclusion of the FN extra domain I (EDI) exon into mature mRNA. Deletion analysis of enhancer subdomains and competitions in vivo with excess of specific enhancer DNA subfragments demonstrate that the "minimal" enhancer, consisting of two 72-bp repeats, is responsible for the splicing effect. The 72-bp repeat region has been reported to promote RNA pol II elongation. When transcription is driven by the alpha-globin promoter linked to the SV40 enhancer, basal EDI inclusion and activation by the SR (Ser-Arg-rich) protein SF2/ASF are much lower than with other promoters. Deletion of only one of the two 72-bp repeats not only provokes higher EDI inclusion levels but allows responsiveness to SF2/ASF. These effects are the consequence of a decrease in RNA pol II elongation evidenced both by an increase in the proportions of shorter proximal over full length transcripts and by higher pol II densities upstream of the alternative exon detected by chromatin immunoprecipitation.

A family of compact plasmid vectors for DNA immunization in humans.

DNA immunization technology is based on the availability of adequate vectors for cloning and expression of heterologous immunoactive proteins in mammalian cells. We have developed a family of DNA plasmid vectors suitable to manipulate antigen expression and location. Their in vitro and in vivo functionality and application are also reported. The developed immune response, the aspects considered for vector design, and the possible independent manipulation of both blocks for the generation of bicistronic constructs, make of the pAEC family of plasmid vectors a source for DNA vaccine candidate's development for further evaluation in human clinical trials, and for potential use in the gene therapy approach.

Cell lineage relationship in the stomach of normal and genetically manipulated mice.

The oxyntic mucosa of the mouse stomach is lined with a heterogeneous population of cells that form numerous short pits continuous with long tubular glands. Tritiated thymidine radioautography has made it possible to pinpoint the origin of all cell types and to follow the differentiation/migration of different cell lineages along the pit-gland unit. The proliferating multipotent stem cells functionally anchored in the upper glandular region, the isthmus, give rise to three main lineage precursors: 1) pre-pit cells, which migrate upward to the pit while differentiating into mucus-producing pit cells; 2) pre-neck cells, which migrate downward to the glandular neck while differentiating into mucus-producing neck cells that, by approaching the glandular base, gradually change their phenotype into pepsinogen- and intrinsic factor-producing zymogenic cells; 3) pre-parietal cells, which differentiate into acid-producing parietal cells in the isthmus and then undergo bipolar migration towards the pit and the glandular base. Thus, parietal cells are the only cells that complete their differentiation in the isthmus and then migrate to be scattered throughout the pit-gland unit. To determine whether parietal cells play a role in controlling decisions about cell fate within the pit-gland unit, the gastric epithelium has been examined in transgenic mice expressing the H,K-ATPase beta-subunit-1035 to +24/simian virus 40 large T antigen fusion gene. The blockade in parietal cell differentiation in these mice produces an amplification of lineage precursors, a marked depletion of zymogenic cells and an increase in pit cell census. Ablation of parietal cells in another transgenic mouse model expressing the H,K-ATPase beta-subunit-1035 to +24/diphtheria toxin fragment A fusion gene also produces amplification of lineage precursors, and similar effects on zymogenic and pit cell census. These findings strongly suggest that parietal cells produce regulatory signals that control the cellular differentiation program of both pit and zymogenic cell lineages, and would hopefully improve our ability to identify the cellular pathways leading to malignant transformation.

Singlet oxygen induced mutation spectrum in mammalian cells.

In order to characterize the molecular nature of singlet oxygen (1O2) induced mutations in mammalian cells, a SV40-based shuttle vector (pi SVPC13) was treated with singlet oxygen arising from the thermal decomposition of the water-soluble endoperoxide of 3,3'-(1,4-naphthylidene) dipropionate (NDPO2). After the passage of damaged plasmid through monkey COS7 cells, the vector was shuffled into E. coli cells, allowing the screening of supF mutants. The mutation spectrum analysis shows that single and multiple base substitutions arose in 82.5% of the mutants, the others being rearrangements. The distribution of mutations within the supF gene is not random and some hotspots are evident. Most of the point mutations (98.4%) involve G:C base pairs and G:C to T:A transversion was the most frequent mutation (50.8%), followed by G:C to C:G transversion (32.8%). These results indicate that mutagenesis in mammalian cells, mediated by 1O2-induced DNA damage, is targeted selectively at guanine residues.

Immunoprecipitation of SV40 replicating minichromosomes complexed with bacteriophage T4 gene 32 protein.

Simian Virus 40 (SV40) DNA replication is a useful model to study eukaryotic cell DNA replication because it encodes only one replication protein and its genome has a nucleoprotein structure ('minichromosome') indistinguishable from cellular chromatin. Late after infection SV40 replicating DNA molecules represent about 5% of total viral minichromosomes. Since gene 32 protein (P32) from bacteriophage T4 interacts with single-stranded DNA and SV40 replication complexes are expected to contain single-stranded regions at the replication forks, we asked whether P32 might be used to isolate replicating SV40 minichromosomes. When nuclear extracts from SV40 infected cells were treated sequentially with P32 and anti-P32 antibodies, pulse-labeled minichromosomes were selectively immunoprecipitated. Agarose gel electrophoresis analysis confirmed that immunoprecipitated material corresponded to SV40 replicative intermediates. Protein analysis of the pelleted material revealed several proteins of viral and cellular origin. Among them, T antigen and histones were found to be complexed with at least other three proteins from cellular origin, to the replicative complexes. Additionally, anti-P32 antibodies were able to detect three cellular proteins of approximately 70, 32 and 13 kDa in western blots. These proteins could correspond to those found as part of an eukaryotic multisubunit single-stranded DNA binding protein. The use of P32 and anti-P32 antibodies thus allows the separation of replicating from mature SV40 minichromosomes and can constitute a novel method to enrich and to study replicative active chromatin.

Singlet oxygen induced DNA damage and mutagenicity in a single-stranded SV40-based shuttle vector.

The effects of singlet oxygen (1O2), generated by the thermal decomposition of water soluble NDPO2 (endoperoxide of the disodium 3,3'-(1,4-naphthylidene) dipropionate), on a single-stranded shuttle vector were analysed. 1O2 induces a much higher level of breaks in the phosphodiester backbone of single-stranded than double-stranded DNA. This may be due to a higher accessibility of guanine residue, primarily damaged by 1O2. The damaged vector was transfected into monkey COS7 cells where single-stranded DNA was converted to the double-stranded replicative form DNA. After 3 days, extrachromosomal DNA was extracted and the plasmids rescued in E. coli to study mutagenesis. There is a significant increase in mutation frequency of damaged single-stranded DNA in comparison to untreated DNA. It is concluded that 1O2 induces breaks in the backbone of single-stranded DNA and that the 1O2-damaged molecules are mutated after passage through mammalian cells.

SV40-based shuttle viruses.

We summarize in this paper the advantages of the shuttle virus system. These SV40-based vectors exhibit the unique properties of being packaged as SV40 pseudo-virions and of being able to infect host cells. Using these transient vectors, we show that their replication can be regulated in some monkey cell lines, in such a way that either low or very high amounts of plasmid DNA can be obtained. The stability of these infectious shuttle vectors in different conditions is analyzed by rescuing them in E. coli, using various gene mutation targets. Moreover, we describe a new series of vectors which can be produced as single-stranded DNA in bacteria. They allow the transfection of a plasmid genome into mammalian cells, as either single-stranded or double-stranded DNA.