Sstr2A: a relevant target for the delivery of genes into human glioblastoma cells using fiber-modified adenoviral vectors.

Glioblastomas are the most aggressive of the brain tumors occurring in adults and children. Currently available chemotherapy prolongs the median survival time of patients by only 4 months. The low efficiency of current treatments is partly owing to the blood-brain barrier, which restricts the penetration of most drugs into the central nervous system. Locoregional treatment strategies thus become mandatory. In this context, viral tools are of great interest for the selective delivery of genes into tumoral cells. Gliomas express high levels of type 2 somatostatin receptors (sstr2A), pinpointing them as suitable targets for the improvement of transduction efficiency in these tumors. We designed a new adenoviral vector based on the introduction of the full-length somatostatin (SRIF (somatotropin release-inhibiting factor)) sequence into the HI loop of the HAdV fiber protein. We demonstrate that (i) HAdV-5-SRIF uptake into cells is mediated by sstr2A, (ii) our vector drives high levels of gene expression in cells expressing endogenous sstr2A, with up to 65-fold enhancement and (iii) low doses of HAdV-5-SRIF are sufficient to infect high-grade human primary glioblastoma cells. Adenoviral vectors targeting SRIF receptors might thus represent a promising therapeutic approach to brain tumors.

Influence of chimeric human-bovine fibers on adenoviral uptake by liver cells and the antiviral immune response.

Human adenoviruses (HAdV) are widely used for in vitro and in vivo gene transfer. Viral hepatotropism, inflammatory responses and neutralization by pre-existing antibodies (NAbs) are obstacles for clinical applications of HAdV vectors. Although the multifactorial events leading to innate HAdV toxicity are far from being elucidated, there is a consensus that the majority of intravenously injected-HAdV vectors is sequestered by Kuppfer cells, probably independently of coagulation factors. In this study, we show that the adenoviral-associated humoral and innate cytokine immune responses are significantly reduced when HAdV-5 vector carrying human bovine chimeric fibers (HAdV-5-F2/BAdV-4) is intravenously injected into mice. Fiber pseudotyping modified its interaction with blood coagulation factors, as FIX and FX no longer mediate the infection of liver cells by HAdV-5-F2/BAdV-4. As a consequence, at early time points post-infection, several cytokines and chemokines (IFN-gamma, IL-6, IP-10, MCP-1, RANTES and MP1beta) were found to be present at lower levels in the plasma of mice that had been intravenously injected with HAdV-5-F2/BAdV-4 compared with mice injected with the parental vector HAdV-5. Moreover, genetic modification of the fiber allowed HAdV-5-F2/BAdV-4 to partially escape neutralization by NAbs.

Non-heparan sulfate GAG-dependent infection of cells using an adenoviral vector with a chimeric fiber conserving its KKTK motif.

Recently, the potential involvement of the putative heparan sulfate proteoglycans (HSPG) binding motif, KKTK, in mediating HAdV-5 liver cell infection following intravascular virus delivery has been debated. In the present study, we demonstrated that HSPGs were not involved in the in vitro infection process of an adenoviral vector harboring chimeric fibers without mutation in the KKTK motif, HAdV-5-F2/BAdV-4. The entry of HAdV-5-F2/BAdV-4 into cells occurs by two mechanisms 1) the attachment of HAdV-5-F2/BAdV-4 to the surface of cells requires N-glycosylation, 2) the uptake of the virus is effective after interaction with a co-receptor, putatively the chondroitin sulfate C. Together, these results contribute to improving our understanding of the molecular mechanisms determining HAdV's infectivity in vitro and may aid in designing novel HAdV-based vectors for gene therapy applications.

Characterization of new mutations in the coding sequence and 5'-untranslated region of the human prostacylcin synthase gene (CYP8A1).

Inheritable interindividual differences in prostacyclin production may be implicated in the pathogenesis of several human vascular diseases. Using a polymerase chain reaction/single-strand conformation polymorphism strategy, we screened for mutations in the gene encoding cytochrome P450 prostacyclin synthase (CYP8A1). DNA samples from healthy French volunteers (n = 130) of Caucasian origin were examined. Five mutations, comprising two previously reported silent mutations and three novel rare missense mutations (P38L, S118R, and R379S), were identified in the coding sequence of the gene. In the 5'-proximal region, we also found a variable number of tandem repeats (VNTR) polymorphism that consisted of four different alleles with 4-6 tandem repeats of a 9-bp unit containing a putative Spl transcriptional factor binding site. One of these (R6), a frequent allele (23.6% of alleles tested) harboring six repeats, is novel, whereas the other three are known. In vitro analysis of the effect of each VNTR allele on promoter activity of a reporter gene was performed by a transient transfection assay. Data confirmed the modulator effect of the VNTR polymorphism on reporter gene transcription. Furthermore, the data demonstrate that allele R6 has the most potent inducing effects in the A549 cell line and, after IL-6 stimulation, in human pulmonary artery endothelial cells. Overall, the data demonstrate that CYP8A1 is polymorphic in Caucasians, and that a polymorphism affecting the 5'-proximal region may result in interindividual differences in CYP8A1 transcriptional regulation in vivo. Additional factors, such as the presence of inflammatory mediators, may be required to modulate transcription of the CYP8A1 gene.

c-Myb protein binds to the EP element of the HBV enhancer and regulates transcription in synergy with NF-M.

The hepatitis B virus (HBV) enhancer contains multiple active elements, one of which is the EP element, a 15 bp site important for its regulation by acting on other functional elements like the E site. The EP element, in the HBV enhancer context, contains two putative binding sites for c-myb family gene products. Electrophoretic mobility shift assays showed that the minimal c-Myb DNA-binding domain binds to the EP sequence. DNase I footprinting experiments revealed that only one consensus binding site was effectively protected. We found that c-Myb down-regulates transcription driving by the HBV enhancer in CAT assays performed in a haematopoietic (K562) and in a hepatic (HepG2) cell line. Interestingly, co-expression of both c-Myb and NF-M, a C/EBPbeta homologue which recognises the E element of the HBV enhancer, showed a synergistic transactivation of the HBV enhancer while, separately, each of them had an inhibitory effect on transcription in HepG2 and K562 cell lines, two cell types potentially infected by the hepatitis B virus.

Characterization of a variable number tandem repeat region in the thiopurine S-methyltransferase gene promoter.

Characterization of the genetic polymorphism of thiopurine S-methyltransferase enzyme (TPMT; EC 2.1.1.67) is required because of its clinical importance for patients exposed to thiopurine drugs. A number of point mutations have already been characterized in exons and introns of the TPMT gene. Here we report the identification of a polymorphic locus within the promoter region of the gene. This polymorphism was detected by polymerase chain reaction - single strand conformation polymorphism analysis of DNA samples from 54 unrelated European individuals. A total of five alleles with length variations were distinguished through the 5'-flanking region involved in the TPMT gene expression. Sequence analysis revealed that these variations were due to a variable number of tandem repeats (VNTR), ranging from four to eight repeats. Each repeat consists of 17 or 18 bp units and contains putative binding sites for transcription factors. The most frequent alleles harbour four or five tandem repeats, a heterozygosity rate of 0.44 was calculated, and a stable Mendelian inheritance of alleles was demonstrated. Analysis of the effect of each VNTR allele on promoter activity of a reporter gene was further performed in various cell lines by transient transfection assay. A modulatory effect of VNTR alleles was observed in vitro, but the repeat polymorphism did not display a significative role in TPMT gene regulation in vivo. Further studies need to be carried out to support the hypothesis that VNTR may contribute to the large interindividual variations of TPMT activity.

The DNA-binding domain of human c-Abl tyrosine kinase promotes the interaction of a HMG chromosomal protein with DNA.

The biological activity of the c-Abl protein is linked to its tyrosine kinase and DNA-binding activities. The protein, which plays a major role in the cell cycle response to DNA damage, interacts preferentially with sequences containing an AAC motif and exhibits a higher affinity for bent or bendable DNA, as is the case with high mobility group (HMG) proteins. We have compared the DNA-binding characteristics of the DNA-binding domain of human c-Abl and the HMG-D protein from Drosophila melanogaster. c-Abl binds tightly to circular DNA molecules and potentiates the interaction of DNA with HMG-D. In addition, we used a series of DNA molecules containing modified bases to determine how the exocyclic groups of DNA influence the binding of the two proteins. Interfering with the 2-amino group of purines affects the binding of the two proteins similarly. Adding a 2-amino group to adenines restricts the access of the proteins to the minor groove, whereas deleting this bulky substituent from guanines facilitates the protein-DNA interaction. In contrast, c-Abl and HMG-D respond very differently to deletion or addition of the 5-methyl group of pyrimidine bases in the major groove. Adding a methyl group to cytosines favours the binding of c-Abl to DNA but inhibits the binding of HMG-D. Conversely, deleting the methyl group from thymines promotes the interaction of the DNA with HMG-D but diminishes its interaction with c-Abl. The enhanced binding of c-Abl to DNA containing 5-methylcytosine residues may result from an increased propensity of the double helix to denature locally coupled with a protein-induced reduction in the base stacking interaction. The results show that c-Abl has unique DNA-binding properties, quite different from those of HMG-D, and suggest an additional role for the protein kinase.

The DNA binding domain of the human c-Abl tyrosine kinase preferentially binds to DNA sequences containing an AAC motif and to distorted DNA structures.

The c-Abl tyrosine kinase protein is implicated in the signaling pathway as well as in transcription, DNA repair, apoptosis, and several other vital biological processes essential for cell proliferation or differentiation. The interaction of c-Abl with DNA is important for some of these functions, but the exact nature of this interaction is still a matter of controversy. The present study addresses the DNA-binding properties of the human c-Abl protein. Using CASTing experiments, the consensus binding site 5'-AA/CAACAAA/C was determined. The central highly conserved AAC triplet appears to constitute the crucial core element in the binding sequences of the c-Abl protein. The c-Abl DNA-binding domain recognizes specific sequences and interacts with deformed DNA structures such as four-way junctions and bubble DNA containing a large single-stranded loop, as determined by electromobility shift assay, melting temperature studies, and binding to specific oligonucleotides covalently linked to beads. Additional competition experiments suggest that the interaction mainly involves contacts within the minor groove of the double helix. The DNA-binding properties of c-Abl are reminiscent of those of high-mobility group (HMG)-like proteins such as LEF-1 and SRY. However, the circular permutation and ring closure assays and DNA unwinding experiments reveal that, unlike HMGs, c-Abl does not bend its target sequence. In addition, it is shown that the protein potentiates the DNA relaxation activity of topoisomerase I. These findings indicate that the interaction of c-Abl with DNA is both sequence-selective and structure-dependent.

Determination of the human c-Abl consensus DNA binding site.

c-Abl tyrosine kinase, an essential protein of the cell cycle signalling pathways, is implicated in the regulation of RNA polymerase II activity, apoptosis and DNA repair. Its DNA binding activity is important for its biological functions. However, the molecular basis of c-Abl interaction with DNA remains largely unclear. We delimited the human c-Abl DNA binding domain and identified its preferred binding site, 5'-A(A/C)AACAA(A/C). The central AAC motif is highly conserved and constitutes the major core element in the binding sites. EMSAs and footprinting experiments were performed to explore how the c-Abl fusion protein recognizes specific sequences in DNA.

Activation of p65 NF-kappaB protein by p210BCR-ABL in a myeloid cell line (P210BCR-ABL activates p65 NF-kappaB).

The chimeric tyrosine kinase p210BCR-ABL is involved in the pathogenesis of chronic myelogenous leukemia. It transforms immature hematopoietic cells in vitro and abrogates IL-3-dependent growth. The mechanisms by which p210BCR-ABL mediates its oncogenicity are not well elucidated. Identifying transcription factors targeted by the chimeric protein may help to clarify these mechanisms. We have analysed the effect of p210BCR-ABL expression on NF-kappaB activity in DA1 cells (an IL-3-dependent murine myeloid progenitor cell line). A specific stimulation of NF-kappaB activity by kinase-active wild-type p210BCR-ABL has been evidenced by transcriptional activation assays. Electrophoretic mobility supershift assays revealed the presence of p65 protein (RelA) DNA binding activity in p210BCR-ABL transformed DA1 cells but not in parental DA1 cells. Activation of RelA in transformed DA1 cells may occur by protein stabilization. Experiments using oligonucleotides antisense to RelA showed that p210BCR-ABL transfected cells failed to survive after IL-3 removal. Moreover, inhibition of cellular growth was shown following treatment of p210BCR-ABL transformed DA1 cells by p65 antisense oligonucleotides. This study suggests that p65 NF-kappaB may be an effector for p210BCR-ABL and probably contributes to its induced transformation process.

Downregulation of major histocompatibility complex class I expression and susceptibility to natural killer cells in cells transformed with the oncogenic adenovirus 12 are regulated by different E1A domains.

All adenoviruses transform rodent cells in vitro, but only cells transformed by serotypes belonging to subgroups A (Ad12) and B (Ad3) are tumorigenic for immunocompetent animals. In these cells, the expression of major histocompatibility complex (MHC) class I antigens is repressed and might allow them to escape from recognition by cytotoxic T lymphocytes and to develop in tumor. Furthermore, these cell lines appear resistant to lysis by natural killer (NK) cells. To determine the E1A domain(s) responsible for these properties several cell lines were created by transforming baby rat kidney cells with a set of plasmids expressing different Ad2/Ad12 hybrid E1A gene products. The class I gene expression was inhibited in cells expressing the Ad12 13S mRNA product and in cells transformed with Ad2/Ad12 hybrid E1A gene product harboring the C-terminal part of the conserved region (CR) 3 of Ad12. Susceptibility of these transformed cell lines to NK cells was determined by cytolytic assays. The results obtained suggest that two of Ad12 E1A domains are required to induce resistance of the cell lines to NK cells.

Susceptibility to natural killer cells and down regulation of MHC class I expression in adenovirus 12 transformed cells are regulated by different E1A domains.

All human adenoviruses transform rodent cells in vitro, but only cells transformed by serotypes belonging to subgroups A (Ad12) and B (Ad3) are tumorigenic for immunocompetent animals. In these cells, the expression of MHC-class I antigens is repressed and might allow them to escape from recognition by cytotoxic T lymphocytes (CTL) and to develop in tumor. Furthermore, these cell lines appear resistant to lysis by natural killer (NK) cells. To determine the E1A domain(s) responsible for these properties several cell lines were created by transforming baby rat kidney (BRK) cells with a set of plasmids expressing different Ad2/Ad12 hybrid E1A gene products. The MHC class 1 gene expression was inhibited in cells expressing the Ad12 13S mRNA product and in cells transformed with Ad2/Ad12 hybrid E1A gene product harboring the C-terminal part of the conserved region (CR) 3 of Ad12. Susceptibility of these transformed cell lines to NK cells was determined by cytolytic assays. The results obtained suggest that two Ad12 E1A domains are required to induce resistance of the cell lines to NK cells.

Comparison between E1A gene from oncogenic and non-oncogenic adenoviruses in cellular transformation (Ad E1A conserved region).

All adenoviruses transform primary BRK cells in vitro, but only cells transformed by oncogenic adenoviruses are tumorigenic for immunocompetent animals. The transforming E1 regions of human Ad 2 and Ad 12 also differ from each other in the frequency in which they can transform BRK cells. We have investigated these properties which can be assigned to the specific domain of the E1A region. For this purpose, chimeric E1A regions between Ad 2 and Ad 12 have been constructed. The efficiency of cell transformation appeared to be determined by the encoding region. The promoter sequences were not important for an efficient cellular transformation although the E1B region cis activated in E1A transcription in both cell transformation and transient expression. We show that sequences located in the E1B promoter were responsible for this effect. In the encoding region the CR 1 domain was essential for the cell transformation frequency.

Adenovirus capsid proteins interact with HSP70 proteins after penetration in human or rodent cells.

Soon after penetration of adenovirus serotype 2 in BHK-21 and HeLa cells, HSP70 and HSC70 proteins become associated with the viral capsid. By analysis with a polyclonal antibody derived from a fusion protein containing the C-terminal domain, 290 amino acids of HSP70, and using both immunological methods and infected cells fractionation we observed that a significant amount of HSP70 proteins moved to the nucleus and colocalized with the adenovirus particles. HSP70 proteins of infected cells were isolated as a complex cross-linked with intracytoplasmic adenovirus type 2. By coprecipitation, using a polyclonal-specific antiserum derived from the fusion protein, or two different monoclonal-specific antisera, we showed that HSP70 and HSC70 proteins were associated with hexon, the major adenovirus capsid protein.

Growth of fastidious adenovirus serotype 40 in HRT 18 cells: interactions with E1A and E1B deletion mutants of subgenus C adenoviruses.

Growth of fastidious adenovirus serotype 40 (Ad40) in several cell lines was investigated. Ad40 was able to readily propagate in human intestinal cell line, HRT 18. Coinfection assays were made in non-permissive and permissive cells between Ad40 and Ad5dl312 or dl1520, mutants deleted in E1A and E1B regions, respectively, to test the ability of Ad40 to complement these mutants and vice versa. Ad40 could enhance Ad5dl312 DNA synthesis in HRT18 and HeLa cells, although its own DNA disappeared in the presence of this mutant in HRT18 cells. In coinfection with dl1520, Ad40 DNA synthesis was inhibited by dl1520 in HRT18 cells and dl1520 DNA synthesis was inhibited by Ad40 in 293 cells. This might reflect the presence of unusual products encoded by Ad40 E1B region.

Regulation of the biosynthesis of subgroup C adenovirus protein IVa2.

The IVa2 gene is located between 16 and 11.3 map units on the left strand of the adenovirus type 5 (Ad5) genome. The coded RNA contains an intron of 277 nucleotides. To determine whether protein IVa2 is synthetized during productive infection and to obtain an immunological reagent to study its function, we prepared antibodies directed to 414 amino acids of protein IVa2 fused to the N-terminal domain of Staphylococcus aureus protein A. Western immunoblot analysis of viral proteins demonstrates that protein IVa2 is a minor component of mature viral particles and that it is also present in assembly intermediates and young virions. Thus, contrary to a previous report (H. Persson, B. Mathisen, L. Philipson, and U. Pettersson, Virology 93:198-208, 1979), protein IVa2 is not related to the 50-kDa polypeptide, a scaffolding protein present in assembly intermediates. The biosynthesis of protein IVa2 during productive infection was examined. Time course studies using immunofluorescence analysis with polyclonal antibodies targeted to protein IVa2 revealed that this protein is first synthesized at 12 h in a few cells exhibiting very striking fluorescence. Synthesis continues until at least 24 h postinfection. When hydroxyurea is added, protein IVa2 is not detected. In cells infected with mutant H5 ts125, blocked at the nonpermissive temperature (40 degrees C) in viral DNA replication, protein IVa2 is overexpressed. These results suggest that protein IVa2 synthesis requires cellular rather than viral DNA replication. RNase protection assay results indicate that hydroxyurea inhibits protein IVa2 synthesis at the transcriptional level. Thus, overexpression of protein IVa2 in H5 ts125-infected cells may be regulated at the translational level.

Nucleotide sequence and regulation of the adenovirus type 3 E2A early promoter.

The nucleotide sequence of the adenovirus serotype 3 E2A early promoter has been determined. In contrast to Ad2, the Ad3 E2A early promoter possessed only one TATA-like box and one nuclear transcription factor E2F binding site and lacked the silencer sequences; however, as in Ad2, the ATF binding site was present. Moreover, the Ad3 E2A promoter harbored a protein binding sequence recognized by the SP1 factor. By transient expression analysis in HeLa cells, we demonstrated that the E1A gene products of Ad3 and Ad2 stimulated Ad3 E2A transcription. In competition experiments, the Ad3 E2A promoter was used in preference to the Ad2 E2A promoter.

Cellular transformation by E1 genes of enteric adenoviruses.

The ability of Ad40 and Ad41 E1A plus E1B genes to transform BRK cells was considerably lower than that of Ad5 and Ad12 corresponding genes. However, as for Ad5, the E1A genes of enteric adenoviruses could cooperate with an activated ras oncogene for full cell transformation and the Ad41 E1B could be complemented by E1A gene of Ad5 or Ad12 for cell transformation. Complementation studies suggested that the conserved region 1 of Ad41 E1A was responsible for this inefficient transformation. The Ad40- and Ad41-transformed cell lines exhibited a low level of major histocompatibility complex (MHC) class I antigens correlated to the low level of Ad12-transformed cells. Class I MHC antigen amounts expressed at the surface of the cells transformed by the weakly oncogenic Ad3 were between the high level of Ad5- and the low level of Ad12-transformed cells.

Adenovirus transformed monkey cell lines permissive to enteric adenovirus type 40.

Plasmids containing the E1 regions of adenovirus serotypes 3 and 5 were transfected into primary Rhesus monkey kidney cells. The presence of viral DNA sequences was detected in transformed cell lines. All these cell lines expressed the E1A proteins. In addition, Ad5 transformed cells, have the E1B 21 kDa protein located in the nuclear membrane. These cell lines were permissive to the enteric adenovirus serotype 40 but not to serotype 41.