Adenovirus-based vascular endothelial growth factor gene delivery to human pancreatic islets.

Islet transplantation is limited by islet graft failure due to poor revascularization, host immune rejection and nonspecific inflammatory response. Delivery of human vascular endothelial growth factor (hVEGF) gene to the islets is likely to promote islet revascularization and survival. We used a bicistronic adenoviral vector encoding hVEGF and CpG-free allele of green fluorescent protein (Adv-GFP-hVEGF) and introduced into human pancreatic islets by transfection. We found that transfection efficiency and apoptosis were dependent on the multiplicity of infection (MOI). Compared to Adv-GFP transfected and nontransfected islets, the levels of hVEGF secreted from Adv-GFP-hVEGF transfected islets were higher and exhibit a linear relationship between hVEGF expression and MOI (10-5000). Persistent, but low level expression of hVEGF from nontransfected islets was also observed. This may be due to expression of the endogenous hVEGF gene under hypoxic conditions. The levels of DNA fragmentation determined by ELISA of islet lysates were dependent on the MOI of Adv-GFP-hVEGF. On glucose challenge, insulin release from transfected islets was comparable to nontransfected islets. Immunohistochemical staining for hVEGF was very high in Adv-GFP-hVEGF transfected islets. Weak staining was also observed for hCD31 in both transfected and nontransfected islets. These findings suggest that Adv-GFP-hVEGF is a potential candidate for promoting islet revascularization.

Chicken Y-box proteins chk-YB-1b and chk-YB-2 repress translation by sequence-specific interaction with single-stranded RNA.

Y-Box proteins comprise a large family of multifunctional proteins with a wide spectrum of activities in both transcription and translational regulation of gene expression. Earlier, we have reported on the involvement of chk-YB-2 in transcriptional regulation of Rous sarcoma virus long terminal repeats and the involvement of chk-YB-1b in transcriptional regulation of alpha1(I) collagen genes. Here, we have investigated the potential role of chk-YB-2 and chk-YB-1b in RNA metabolism. We report that chk-YB-2 and chk-YB-1b are localized predominantly in the cytoplasm and that they both can bind single-stranded RNA in a sequence-specific and reversible manner. Well-conserved cold-shock domain, N-terminal proline-rich domain and the alternating clusters of acidic and basic amino acids located in the C-terminal ends of these two proteins were all found to be necessary for their RNA-binding ability. Further, we demonstrate that these two proteins inhibit translation in vitro and that binding to RNA is required for this inhibition. The significance of these results is discussed.

Angiotensin II and extracellular matrix homeostasis.

As a circulating hormone, endocrine properties of angiotensin (Ang) II are integral to circulatory homeostasis. Produced de novo its autocrine/paracrine properties contribute to biologic responses involving various connective tissues (e.g. extracellular matrix, adipose tissue, bone and its marrow). In this brief review, we develop the concept of extracellular matrix homeostasis, a self regulation of cellular composition and structure, wherein fibroblast-derived AngII regulates elaboration of TGF-beta 1, a fibrogenic cytokine responsible for connective tissue formation at normal and pathologic sites of collagen turnover.

Inhibition of the rous sarcoma virus long terminal repeat-driven transcription by in vitro methylation: different sensitivity in permissive chicken cells versus mammalian cells.

Rous sarcoma virus (RSV) enhancer sequences in the long terminal repeat (LTR) have previously been shown to be sensitive to CpG methylation. We report further that the high density methylation of the RSV LTR-driven chloramphenicol acetyltransferase reporter is needed for full transcriptional inhibition in chicken embryo fibroblasts and for suppression of tumorigenicity of the RSV proviral DNA in chickens. In nonpermissive mammalian cells, however, the low density methylation is sufficient for full inhibition. The time course of inhibition differs strikingly in avian and mammalian cells: although immediately inhibited in mammalian cells, the methylated RSV LTR-driven reporter is fully inhibited with a significant delay after transfection in avian cells. Moreover, transcriptional inhibition can be overridden by transfection with a high dose of the methylated reporter plasmid in chicken cells but not in hamster cells. The LTR, v-src, LTR proviral DNA is easily capable of inducing sarcomas in chickens but not in hamsters. In contrast, Moloney murine leukemia virus LTR-driven v-src induces sarcomas in hamsters with high incidence. Therefore, the repression of integrated RSV proviruses in rodent cells is directed against the LTR.

Triple helix formation with the promoter of human alpha1(I) procollagen gene by an antiparallel triplex-forming oligodeoxyribonucleotide.

The promoters of alpha1(I) procollagen genes of vertebrates contain two contiguous stretches of polypyrimidine/polypurine sequences, referred to as C1 (-140 to -170) and C2 (-171 to -200). Antiparallel triplex-forming upstream oligonucleotides form efficient triplexes with C1. The C1 tract of human differs from rodent alpha1(I) promoters by 7 nt which are mainly A-->G transitions. Human triplex-forming oligodeoxyribonucleotide (TFO) formed stable triplexes efficiently with a K d of approximately 10-20 nM compared with a K d of approximately 100 nM for rodent TFO. Mutational analysis indicated that 3 or 4 nt (-153 to -155) are sufficient for this higher affinity. TFOs specific for human C1 inhibited transcription from human promoter both in vitro in HeLa cell nuclear extracts and in vivo in cultured chick embryo fibroblasts.

chk-YB-1b, a Y-box binding protein activates transcription from rat alpha1(I) procollagen gene promoter.

Type-I collagen, the predominant component of extracellular matrix, is a triple-helical protein consisting of two alpha1 polypeptides and one alpha2 polypeptide. Expression of alpha1 and alpha2 procollagen genes is co-ordinately regulated under both normal and various pathological conditions. However, the basis of this co-ordinate regulation is not well known. YB-1b, a Y-box protein, has been shown to bind to the polypyrimidine tract present in the alpha2 procollagen gene. Here, we show that chk-YB-1b, a YB-1 homologue, binds in a single-strand-sequence-specific manner to the highly conserved pyrimidine-rich sequences in both alpha1(I) and alpha2(I) procollagen promoters from different species, as demonstrated by electrophoretic-mobility-shift assays and by DNaseI footprinting experiments. Transiently transfected and retrovirally expressed antisense oligonucleotides directed against chk-YB-1b specifically inhibited the alpha1(I) procollagen promoter-driven transcription in cultured fibroblasts. Considering these data and the fact that the chk-YB-1b binding site is one of the few sites between alpha1(I) and alpha2(I) procollagen promoters that is conserved from chicken to human, it is proposed that chk-YB-1b may be involved in co-ordinate expression of these two collagen genes.

Retroviral vectors for gene therapy.

Although there are many hurdles to overcome for successful gene therapy, there is a vast potential to permanently incorporate genes into cells to correct genetic disorders and to combat viral infections. Retroviruses, inspite of some limitations, offer the best hope in this direction and lentiviral vectors, which infect nondividing cells, may be the choice in the future, especially in gene therapy for central nervous system disorders.

An RNA oligonucleotide corresponding to the polypyrimidine region of the rat alpha 1(I) procollagen promoter forms a stable triplex and inhibits transcription.

In this report we demonstrate formation of a triplex structure by an antiparallel RNA oligonucleotide corresponding to the 21 bp polypurine-pyrimidine stretch from -141 to -162 of the rat alpha 1(I) procollagen promoter with a Kd of 0.1-0.2 microM. The formation of triplexes by the triplex forming oligoribonucleotide (ORN) was also observed under physiological conditions. In vitro transcription run-off experiments showed that triplex formation results in inhibited transcription from the rat alpha 1(I) procollagen gene. Our results demonstrate a novel approach for down-regulation of procollagen gene transcription in vivo.

Role of single-stranded DNA regions and Y-box proteins in transcriptional regulation of viral and cellular genes.

Single-stranded regions, known to be important for optimal rates of transcription, have been observed in the promoters of several cellular genes as well as in the promoters of many pathogenic viruses. Several host-encoded, single-stranded DNA binding proteins capable of binding these regions have been purified and their genes isolated. In this review, information available about single-stranded regions present within various promoters and the interaction of a novel class of single-stranded DNA binding transcription factors belonging to the Y-box family of proteins is reviewed. Mechanisms by which these proteins influence transcription of both cellular and viral genes are proposed.

Characterization of the DNA-binding domain of the avian Y-box protein, chkYB-2, and mutational analysis of its single-strand binding motif in the Rous sarcoma virus enhancer.

chkYB-2 is a sequence-specific, single-stranded DNA binding chicken Y-box protein that promotes Rous sarcoma virus long terminal repeat (RSV LTR)-driven transcription in avian fibroblasts. The DNA-binding domain of chkYB-2 has been mapped by characterizing the DNA binding properties of purified recombinant chkYB-2 mutant polypeptides. The data indicate that the invariant cold shock domain (CSD) is necessary but not sufficient for association with DNA and suggest that another conserved region, adjacent to the carboxyl boundary of the CSD, plays a role in high-affinity DNA binding. chkYB-2 binds to a tandem repeat of the 5'-GTACCACC-3' motif on the RSV LTR. Mutational analysis of this recognition sequence revealed the requirement of an essentially unaltered template for both high-affinity binding by chkYB-2 as well as maximal transcriptional activity of the RSV LTR in vivo. The single-stranded DNA binding activity of chkYB-2 is augmented by Mg2+. The possible significance of this finding for transactivation by a single-strand DNA binding protein is discussed.

Cloning of a novel Y-box homology protein (chkYB-1HP) cDNA lacking the cold-shock domain.

We have isolated the complete cDNA clone of a novel 262 amino acid Chicken YB-1 Homology Protein (chkYB-1HP) by screening a chicken embryo cDNA expression library. While the chkYB-1HP is identical over its carboxyl-terminal 78 amino acids with the Y-box protein YB-1, it differs strikingly from all other Y-box transcription factors by lacking the cold-shock domain (CSD). We propose that proteins like chkYB-1HP that lack the CSD, but retain the hydrophilic carboxyl domain could regulate Y-box proteins through the formation of heterodimeric complexes.

Antiparallel polypurine phosphorothioate oligonucleotides form stable triplexes with the rat alpha1(I) collagen gene promoter and inhibit transcription in cultured rat fibroblasts.

The rat alpha1(I) collagen promoter contains a unique polypurine-polypyrimidine sequence between -141 and -200 upstream of the transcription start site. The polypurine sequence from -171 to -200 (C2) is on the coding strand and the adjacent polypurine sequence from -141 to -170 (C1) is on the non-coding strand. Earlier we demonstrated triplex formation with a polypurine 30 nt parallel triplex-forming oligonucleotide (TFO) corresponding to C1 and inhibition of transcriptional activity of the rat alpha1(I) collagen promoter. In the present work we have tested triplex-forming abilities of shorter (18 nt) purine and pyrimidine TFOs in parallel and antiparallel orientation to the C1 purine sequence. Our results show that purine antiparallel TFOs formed triplexes with the highest binding affinities, while pyrimidine oligodeoxyribonucleotides (ODNs) did not show appreciable binding. Phosphorothioate modification of purine TFOs did not significantly reduce binding affinity. We also demonstrate that preformed triplexes are quite stable when precipitated with ethanol and resuspended in water. Further analysis was carried out using two purine phosphorothioate antiparallel TFOs, 158 APS and 164 APS, designed to bind to the promoter region from -141 to -158 and -147 to -164, respectively, which were found to form triplexes even under physiological conditions. DNase I footprinting experiments showed the ability of these TFOs to protect target sequences in the promoter region; both purine sequences (C1 and C2) were protected in the case of 158 APS. Transfection experiments using preformed triplexes with a reporter plasmid containing the collagen promoter sequence showed significant inhibition of transcription when compared with a control phosphorothioate ODN. The effect of 164 APS was greater than that of 158 APS. These results indicate that this triplex strategy could be used in the down-regulation of collagen synthesis in cultured cells and offer the potential to control fibrosis in vivo.

Molecular cloning and characterization of a cDNA for the highly conserved HMG-like protein (Pf16) gene of Plasmodium falciparum.

A cDNA clone (PfHB3-2-4) of 1538 bp corresponding to the highly conserved HMG-like protein (Pf16) was isolated. However, northern analysis suggests that the mRNA is about 2.2 to 2.3 kb. Analysis by RT-PCR indicated that the 0.6 to 0.7 kb sequence missing in the cDNA maps to the 3' end, suggesting that the cDNA is terminated within the 26 adenosine residues that are in the middle of the Pf16 sequence. The most unique feature about this cDNA is the presence of two open reading frames (ORF), one from nucleotides 91 to 927 and the other starting from 1421. The second ORF corresponds to Pf16. Expression of the cDNA clones in Escherichia coli and translation in rabbit reticulocytes of RNA transcribed from the T7 promoter of the cDNA clones revealed that only the 3' end Pf16 is translated from this mRNA. Further experiments with antisense oligonucleotides specific for Pf16 indicated that the Pf16 protein serves an important function in the life cycle of the parasite.

Chicken YB-2, a Y-box protein, is a potent activator of Rous sarcoma virus long terminal repeat-driven transcription in avian fibroblasts.

We have previously reported on the cloning and characterization of chk-YB-2, a novel member of the Y-box family of proteins, that binds to the sequence 5'-GTACCACC-3' present on the noncoding strand of the Rous sarcoma virus (RSV) long terminal repeat (LTR) in a single-strand-specific manner. Here, we demonstrate that deletion or mutation of this motif not only eliminates chk-YB-2 binding in vitro but also down-regulates RSV LTR-driven transcription in avian cells. Selective abrogation of chk-YB-2 expression by using antisense oligonucleotides decreased RSV LTR-driven transcription in a promoter-specific manner. This inhibition was not observed when a reporter construct with a deletion in the chk-YB-2 binding site was used. Depletion of cellular chk-YB-2 by transfecting the cells with excess of its recognition sequence oligonucleotides also resulted in reduced transcription from the RSV LTR. Taken together, these results suggest that chk-YB-2 acts as an activator of LTR-promoted transcription in avian cells and that this activation is mediated primarily through the sequence 5'-GTACCACC-3'.

The GT-rich sequence in the U5 region of Rous sarcoma virus long terminal repeat is required for transcription termination and 3' processing.

The sequences in the LTR of Rous sarcoma virus that are required for transcription termination and polyadenylation have been determined. A vector containing LTR-neo-LTR has been constructed and deletions in the U5 region of the downstream LTR have been made. The DNAs from wild-type and deletion mutant recombinant plasmids were introduced into QT6 cells and G418-resistant transformants were selected. Those transformants with neo sequences in the arrangement, LTR-neo-LTR, were analyzed for transcription termination and polyadenylation by Northern blot analysis and by S1 protection experiments. The results indicate that the polyadenylation signal, AATAAA, located in the U3 region alone, is not sufficient for 3' end processing and that the sequence between +20 and +44 in the U5 region is absolutely required for transcription termination or endonucleolytic cleavage and polyadenylation.

Identification of negative and positive regulatory elements in the rat alpha 1(I) collagen gene promoter.

Type I collagen is the main constituent of extracellular matrix found in various organs including the heart. Under some pathological conditions accumulation of excess type I collagen in the interstitium leads to organ dysfunction. In order to identify the regulatory elements in the rat alpha 1(I) collagen gene promoter, deletions were made in the promoter region. Various plasmid constructs were transfected into different fibroblasts using LipofectAMINE. The results indicated a negative cis-element between nucleotides -310 to -440 in the rat alpha 1(I) collagen gene promoter. Presence of this sequence significantly diminished the reporter gene activity. In addition we have observed that the sequence between -220 to -330 contained a positively acting cis-element, which is highly active in rat fibroblasts. Analysis of the nuclear factors binding to the negative element by electrophoretic mobility shift assays indicated that similar or identical factors are present in different fibroblasts as well as human HeLa cells and that these factors appear to bind to a composite sequence within -325 to -400. Competition with different oligonucleotides suggested that two distinct but contiguous sequence motifs may constitute the negative regulatory element. Our results with the rat alpha 1(I) collagen promoter confirm the presence of a negative cis-element previously described for the mouse promoter and provided additional information on the bipartite nature of this element.

Cloning of Rous sarcoma virus enhancer factor genes. II. RSV-EF-II, abundantly expressed in fibroblasts and muscle tissue, binds to an octamer sequence, 5'-GTACCACC-3', in the noncoding strand of RSV enhancer.

Rous sarcoma virus (RSV) mainly replicates in avian fibroblasts, and the U3 enhancer region of the long terminal repeats of RSV contains the determinants for its tissue-tropic expression. We describe the cloning and characterization of an avian gene that encodes a protein capable of binding to the enhancer region of Rous sarcoma virus. A PCR-derived probe corresponding to the U3 region of RSV was used to isolate a cDNA clone by screening a chicken cDNA expression library. The cDNA is predicted to encode a polypeptide of 298 amino acids that is homologous to the Y-box (inverted CCAAT) family of DNA-binding transcription factors. This factor, which we refer to as Rous sarcoma virus enhancer factor-II (RSV-EF-II), shows 99% aa identity over a 105-amino-acid stretch that is highly conserved in all Y-box proteins, and is commonly referred to as the cold shock domain. RSV-EF-II selectively binds to single-stranded DNA, and the binding site, as determined by electrophoretic mobility shift assays, consists of the sequence 5' GTACCACC 3' located between nucleotides -112 to -119 in the noncoding strand of the RSV enhancer. Although RSV-EF-II shares considerable homology with the Y-box family of proteins, it does not bind to the inverted CCAAT boxes at positions -65 to -69 and -129 to -133 in the RSV LTR. Northern analysis indicates that RSV-EF-II-specific transcripts are expressed predominantly in avian fibroblasts and muscle tissue. The results of these binding and mRNA expression expriments suggest that RSV-EF-II may play an important role in tissue- and host-specific expression of RSV LTR-driven gene expression. Further, we show that RSV-EF-II acts as a repressor of transcription.

Triple helix-forming oligonucleotide corresponding to the polypyrimidine sequence in the rat alpha 1(I) collagen promoter specifically inhibits factor binding and transcription.

Type I and III fibrillar collagens are the major structural proteins of the extracellular matrix found in various organs including the myocardium. Abnormal and progressive accumulation of fibrillar type I collagen in the interstitial spaces compromises organ function and therefore, the study of transcriptional regulation of this gene and specific targeting of its expression is of major interest. Transient transfection of adult cardiac fibroblasts indicate that the polypurine-polypyrimidine sequence of alpha 1(I) collagen promoter between nucleotides - 200 and -140 represents an overall positive regulatory element. DNase I footprinting and electrophoretic mobility shift assays suggest that multiple factors bind to different elements of this promoter region. We further demonstrate that the unique polypyrimidine sequence between -172 and -138 of the promoter represents a suitable target for a single-stranded polypurine oligonucleotide (TFO) to form a triple helix DNA structure. Modified electrophoretic mobility shift assays show that this TFO specifically inhibits the protein-DNA interaction within the target region. In vitro transcription assays and transient transfection experiments demonstrate that the transcriptional activity of the promoter is inhibited by this oligonucleotide. We propose that TFOs represent a therapeutic potential to specifically influence the expression of alpha 1(I) collagen gene in various disease states where abnormal type I collagen accumulation is known to occur.

Regulation of collagen degradation in the rat myocardium after infarction.

Fibrillar collagens, essential for maintaining the structural integrity of the myocardium, are degraded by matrix metalloproteinase (MMP-1). In other tissues collagenolysis is an important component of wound healing. Here we examined collagen degradation in the myocardium after infarction. Collagenase activity, measured by zymography, and expression of matrix metalloproteinase (MMP-1) and tissue inhibitor of metalloproteinase (TIMP) mRNA, detected by Northern blotting and in situ hybridization, in the rat heart 6 h to 28 days after left coronary artery ligation were studied. Sham-operated rats served as controls. Infarcted left ventricle was compared to non-infarcted right ventricle and interventricular septum and to sham-operated tissues. We found a transient increase in collagenase activity in the infarcted left ventricle, which began at day 2 (4.5-fold increase compared to controls), peaked at day seven (6.5-fold increase) and declined thereafter, together with a concomitant increase and contribution in collagenolytic activity of gelatinases (MMP-2 and MMP-9). An increase in collagenase mRNA was not seen until day 7 and only in the infarcted ventricle, while changes in MMP-1 activity or mRNA expression were not observed at remote sites or in sham-operated controls. Transcription of TIMP mRNA was observed at 6 h (two-fold increase) in the infarcted ventricle, peaked on day two after MI (eight-fold increase) and slowly decreased thereafter. No change in TIMP mRNA expression was observed at remote sites or in sham-operated controls. Cells responsible for transcription of MMP-1 and TIMP mRNA were fibroblast-like cells, not inflammatory or endothelial cells. At the site of infarction post-translational activation of latent collagenase (MMP-1) plays a greater role in the wound healing response than transcription of collagenase mRNA. Collagenase mRNA is synthesized when the latent extracellular pool of MMP-1 is reduced through the activation of latent collagenases and gelatinases. TIMP mRNA synthesis is regulated by the activation of MMPs with the balance between collagenase activation and TIMP inhibition determining the amount of collagenolysis in infarcted tissue.

Cloning of Rous sarcoma virus enhancer factor genes. I. Evidence that RSV-EF-I is related to Y-box (inverted CCAAT) binding proteins and binds to multiple motifs in the RSV enhancer.

A cDNA clone was isolated from a chicken embryo cDNA library employing a PCR-generated radiolabeled probe specific for the U3 region of the Rous sarcoma virus LTR. The cDNA encodes a protein of 345 aa and is homologous to the Y-box (inverted CCAAT) binding proteins. The amino acid sequence of the RSV-EF-I shows 75% identity with rat EF1 but the NH2-terminal 60-aa residues share little homology. At the carboxyl terminus an additional 28-aa sequence, rich in basic residues, probably encoded by an extra exon, is present in the chicken RSV-EF-1. Electrophoretic mobility assays carried out with various radiolabeled oligonucleotides spanning the U3 region of the RSV LTR (-234 to -54) indicated that the RSV-EF-I binds strongly to the sequence AAGGTGGTAC and somewhat less efficiently to the sequences AAGGAAAG and CTTATGCAA. In contrast to rat EF1A which binds to the inverted CCAAT box, RSV-EF-I does not bind to the CCAAT box sequence. These results suggest that the RSV-EF-I, although structurally similar to the rat EF1A, binds differently to more than one cis-acting element. The gene for RSV-EF-I is expressed in a variety of cell lines, although most abundantly in avian fibroblasts compared to mammalian cells. It is barely expressed in normal lives but expressed at significantly enhanced levels in many immortalized hepatocytes and hepatic tumors.