Growth inhibition of cancer cells by co-transfection of diphtheria toxin A-chain gene plasmid with bovine leukemia virus-tax expression vector.

We constructed a plasmid containing bovine leukemia virus (BLV)-tax gene driven by SR alpha promoter, designated as pME-BLVtax, to activate the promoter of the long terminal repeat (LTR) of BLV in various tumor cells. Activation of the promoter of BLV-LTR by pME-BLVtax was confirmed by luciferase assay. When the cells, such as COS-1, C8, and KU-1, were transfected with a plasmid pBLV-LUC1, which contained the luciferase gene under the control of BLV-LTR, and pME-BLVtax, luciferase was expressed in these cells, whereas no luciferase gene expression was observed when only pBLV-LUC1 was introduced into the cells. Activation of the BLV-LTR promoter was regulated by pME-BLVtax and 0.5 microg of pME-BLVtax was sufficient for the expression of the gene under the control of BLV-LTR. Furthermore, pME-BLVtax was used to direct the cell expression of the gene for diphtheria toxin A-chain under the control of BLV-LTR (pLTR-DT) to various tumor cell lines, KU-1, C8, COS-1, BL2M3, and HeLa cells. The transfection was carried out with cationic liposomes. In this experiment, co-transfection of pLTR-DT with pME-BLVtax exerted selective growth inhibitory effects on the tumor cell lines. Moreover, three co-introductions of pLTR-DT with pME-BLVtax into the cell lines resulted in significant inhibition of the cell growth. This result suggests that the delivery of the pLTR-DT and pME-BLVtax genes into tumor cells by the use of cationic liposomes may be potentially useful as a novel approach for the treatment of tumor cells.

Preparative-scale Enzyme-catalyzed Synthesis of (R)-α-Fluorophenylacetic Acid.

A preparative-scale asymmetric synthesis of (R)-α-fluorophenylacetic acid, a useful chiral derivatizing reagent, is described. Starting from ethyl α-bromophenylacetate, α-fluorophenylmalonic acid dipotassium salt was prepared in three steps (54% yield), including nucleophilic substitution by the fluoride ion as the keystep. Both the purified form and crude preparation of arylmalonate decarboxylase in E. coli worked well on this substrate, and (R)-α-flurophenylacetic acid (>99% e.e.) was prepared in a quantitative yield.

Antitumor effect of diphtheria toxin A-chain gene-containing cationic liposomes conjugated with monoclonal antibody directed to tumor-associated antigen of bovine leukemia cells.

Monoclonal antibody c143 against tumor-associated antigen (TAA) expressed on bovine leukemia cells was conjugated to cationic liposomes carrying a plasmid pLTR-DT which contained a gene for diphtheria toxin A-chain (DT-A) under the control of the long terminal repeat (LTR) of bovine leukemia virus (BLV) in the multicloning site of pUC-18. The specificity and antitumor effects of the conjugates were examined in vitro and in vivo using TAA-positive bovine B-cell lymphoma line as the target tumor. In vitro studies with the TAA-positive cell line indicated that luciferase gene-containing cationic liposomes associated with the c143 anti-TAA monoclonal antibody caused about 2-fold increase in luciferase activity compared with cationic liposomes having no antibody, and also that the c143-conjugated cationic liposomes containing pLTR-DT exerted selective growth-inhibitory effects on the TAA-positive B-cell line. Three injections of pLTR-DT-containing cationic liposomes coupled with c143 into tumor-bearing nude mice resulted in significant inhibition of the tumor growth. The antitumor potency of the c143-conjugated cationic liposomes containing pLTR-DT was far greater than that of normal mouse IgG-coupled cationic liposomes containing pLTR-DT as assessed in terms of tumor size. These results suggest that cationic liposomes bearing c143 are an efficient transfection reagent for BLV-infected B-cells lymphoma cells, and that the delivery of the pLTR-DT gene into BLV-infected B-cells by the use of such liposomes may become a useful technique for gene therapy of bovine leukosis.

In vivo antitumor effect of cationic liposomes containing diphtheria toxin A-chain gene on cells infected with bovine leukemia virus.

A plasmid pLTR-DT which contained a gene for diphtheria toxin A-chain (DT-A) under the control of the long terminal repeat (LTR) of bovine leukemia virus (BLV) (BLV-LTR) in the multicloning site of pUC-18 was entrapped in cationic liposomes composed of N-(alpha-trimethylammonioacetyl)-didodecyl-D-glutamate chloride (TMAG), dioleoyl phosphatidylethanolamine (DOPE) and dilauroyl phosphatidylcholine (DLPC) (1:2:2, molar ratio) (TMAG-liposome), and their antitumor effect on BLV-infected tumor cells was examined in vivo. The cationic TMAG-liposome containing pLTR-DT was successively injected into the tumor transplanted to nude mice. The growth of tumor was significantly inhibited by the injection of cationic TMAG-liposome containing pLTR-DT. On the other hand, TMAG-liposome containing pUC18 plasmids showed no such effect. These results suggest that a DT-A expression plasmid under the control of BLV-LTR is highly toxic to the BLV-infected tumor cells, and that the cationic liposomes, such as TMAG-liposome, may be efficient transfection reagent for BLV-infected tumor cells and can be utilized for DT-A gene delivery into BLV-infected tumor cells in vivo.

Evaluation of cationic liposomes for delivery of diphtheria toxin A-chain gene to cells infected with bovine leukemia virus.

We investigated whether cationic liposomes are efficient at delivering the gene for diphtheria toxin A-chain (DT-A) under the control of the long terminal repeat (LTR) of bovine leukemia virus (BLV) (pLTR-DT) into BLV-infected cells and are also suitable for in vivo use. The transfection activity of the cationic liposomes composed of N-(alpha-trimethylammonioacetyl)-didodecyl-D-glutamate chloride (TMAG), dioleoyl phosphatidylethanolamine (DOPE) and dilauroyl phosphatidylcholine (DLPC) (1:2:2, molar ratio) (TMAG-liposome) and liposomes composed of phosphatidylserine (PS) (PS-liposome) was evaluated by the luciferase assay using a plasmid which contains the coding sequence of firefly luciferase under the control of the SR alpha promoter (pSR alpha/L-A delta 5). The TMAG-liposome gave highly efficient transfection in the presence of serum. On the other hand, PS-liposome showed inferior efficiency. When BLV-infected cells were co-transfected with a fixed amount of pSR alpha/L-A delta 5-entrapped TMAG-liposome and various amount of pLTR-DT-containing TMAG-liposome, the luciferase activity in the BLV-infected cells was inhibited by the addition of pLTR-DT-entrapped TMAG-liposome dose-dependently. The cationic TMAG-liposome containing pLTR-DT was successively added to BLV-infected cells in culture. The number of viable cells was markedly reduced by the cationic TMAG-liposome containing pLTR-DT. On the other hand, TMAG-liposome containing pSR alpha/L-A delta 5 showed no such effect. pLTR-DT entrapped by the cationic TMAG-liposome was not digested by the treatment with DNase I and with serum. These results suggest that the cationic liposomes, such as TMAG-liposome, may be efficient transfection reagent for the BLV-infected cells and can be utilized for DT-A gene delivery into the BLV-infected cells in vivo.

Evaluation of an antisense RNA transgene for inhibiting growth hormone gene expression in transgenic rats.

We compared the levels of growth hormone (GH) mRNA in the pituitary, plasma GH concentration, and altered phenotype in rats heterozygous and homozygous for an antisense RNA transgene targeted to the rat GH gene, with those in nontransgenic rats. We initially investigated whether the transgene promoter, which is connected to four copies of a thyroid hormone response element (TRE) that increases promoter activity, affected in vivo transgene expression in the pituitary of the transgenic rats. Plasma GH concentration correlated negatively with T3 injection in surgically thyroidectomized heterozygous transgenic rats. There was a reduction of about approximately 35-40% in GH mRNA levels in the pituitary of homozygous animals compared with those in non-transgenic rats. Plasma GH concentration was significantly approximately 25-32 and approximately 29-41% lower in heterozygous and homozygous transgenic rats, respectively, compared with that in nontransgenic animals. Furthermore, the growth rates in homozygous transgenic rats were reduced by approximately 72-81 and approximately 51-70% compared with those of their heterozygous and nontransgenic littermates, respectively. The results of these studies suggested that the biological effect of GH in vivo is modulated dose-dependently by the antisense RNA transgene. The rat GH gene can therefore be targeted by antisense RNA produced from a transgene, as reflected in the protein and RNA levels.

Growth retardation in rats whose growth hormone gene expression was suppressed by antisense RNA transgene.

We produced four transgenic founder rats (F0) by introducing into rat embryos a fusion gene, which consisted of rat growth hormone (GH) promoter containing with four copies of thyroid hormone response element (TRE) and antisense cDNA sequences for rat GH. This transgene promoter directed 2.8-fold stimulation of CAT gene expression in transfected GH3 rat pituitary tumor cells compared with the rat GH promoter alone. Two of four transgenic rats expressed antisense RNA in the pituitary. Transgenic offspring (F1) from each founder rat exhibited dwarfism at as early as 3-4 weeks of age, and they exhibited approximately 70-85% reduced growth rate compared with their nontransgenic littermates over 56 weeks of observation. Plasma rat GH concentration was approximately 40-50% lower in transgenic F1 rats compared to their nontransgenic littermates. In these experiments, the pituitary hormone expression controlled in a complex manner was shown to be repressed by the antisense RNA transgene. Furthermore, the suppression of gene expression could be achieved by antisense RNA transgene in the rat as well.

Suppressive effect of liposomes containing DNA coding for diphtheria toxin A-chain on cells transformed with bovine leukemia virus.

A recombinant plasmid which contained a gene for diphtheria toxin A-chain (DT-A) under the control of the long terminal repeat (LTR) of bovine leukemia virus (BLV) (BLV-LTR) was constructed to test a novel application of liposomes as antiviral agents. The promoter activity of BLV-LTR was estimated by the chloramphenicol acetyltransferase (CAT) assay using a plasmid which contains the coding sequence of CAT under the control of BLV-LTR (pBLVCAT). When BLV-infected cells were transfected with pBLVCAT, CAT activity was detected. BLV-uninfected cell lines, however, showed no detectable CAT activity. The plasmid DNA entrapped in liposomes was added to BLV-infected cells in culture. Syncytium formation induced by BLV-infected cells was effectively suppressed by the liposomes containing the gene for DT-A under the control of BLV-LTR. Conversely, liposomes containing the gene for DT-A without a promoter showed no such effect. DT-A gene-containing liposomes with BLV-LTR did not affect formation of syncytium induced by bovine immunodeficiency virus. These observations indicate that BLV-infected cells were readily targeted on the level of gene expression. This strategy could be applied to the treatment of BLV-induced B-cell proliferation of cattle, and further to other viral/neoplastic diseases where specific gene expression is exerted.

An amino-terminal fragment of GAL4 binds DNA as a dimer.

GAL4 is a yeast transcriptional activator protein that binds to specific 2-fold rotationally symmetric sites on DNA and stimulates transcription of the genes required for galactose catabolism. The DNA binding region of the protein is located within the first 74 amino acids and contains a "zinc finger" sequence motif. We show that a polypeptide comprising the first 147 amino acids of GAL4, designated GAL4 (1-147), binds DNA as a dimer in vitro. Although a protein containing only the first 74 amino acids, designated GAL4 (1-74), binds DNA specifically, its affinity is reduced relative to GAL4 (1-147). Addition of the strong dimerization domain of lambda repressor to GAL4 (1-74) generates a protein that binds as tightly as GAL4 (1-147). GAL4 (1-147) makes rotationally symmetric contacts with its recognition site when assayed by DNase I, exonuclease III and hydroxyl radical footprinting and by phosphate ethylation interference. Binding of GAL4 (1-147) in vitro requires either zinc or cadmium.

Mechanism of action of a yeast activator: direct effect of GAL4 derivatives on mammalian TFIID-promoter interactions.

We have analyzed interactions between the mammalian TATA factor (TFIID) and derivatives of the yeast activator GAL4. The interaction of the TATA factor on the adenovirus E4 promoter with GAL4 binding sites adjacent to the TATA site was qualitatively altered in response to GAL4 binding. Alterations in the TFIID interactions were observed with two GAL4 derivatives that stimulated hybrid E4 promoter activity in vitro but not with a third derivative that bound to DNA but showed no activation. These results indicate that TFIID is a direct target for a GAL4 activation domain and suggest a simple general model for the activation mechanism.

GAL4 activates gene expression in mammalian cells.

GAL4, a protein that activates transcription in yeast, is shown to activate the mouse mammary tumor virus promoter in mammalian cells. Activation depends upon a GAL4-binding sequence inserted upstream of the gene. Deletion mutants of GAL4 bearing one or both of the "activating regions" required for activation in yeast also activate transcription in mammalian cells. A derivative of GAL4 that binds to DNA but cannot activate transcription in yeast also fails to activate transcription in mammalian cells. We also show that GAL4 and the glucocorticoid receptor activate the mouse mammary tumor virus promoter synergistically.

A putative Ca2+-binding protein: structure of the light subunit of porcine calpain elucidated by molecular cloning and protein sequence analysis.

cDNA clones specific for the light subunit of porcine calpain I have been isolated from a porcine kidney cDNA library. The complete primary structure of the light subunit has been revealed by nucleotide sequence analysis of the cDNA clones isolated and amino acid sequence analysis of peptides isolated from the purified mature protein. We found that the light subunit contains two distinct domains. Domain I, the amino-terminal half, has two unusually long, paired polyglycyl sequences and may serve as a binding site to the heavy subunit. Domain II, the carboxyl-terminal half, is a region highly homologous to the putative Ca2+-binding domain of the heavy subunit of chicken calpain elucidated recently. This region has four potential Ca2+-binding sites, each having the "E-F hand" structure. Our results suggest that the Ca2+-mediated proteolytic activity of calpain is controlled through the cooperative and/or sequential actions of multiple Ca2+-binding sites present in both two-subunit molecules, heavy and light subunits of calpain.

Isolation and structural organization of the human preproenkephalin B gene.

The primary structure of porcine preproenkephalin B has been elucidated by cloning and sequencing cDNA: it contains neoendorphin, dynorphin and leumorphin (containing rimorphin as its amino-terminus). These opioid peptides, each having a leucine-enkephalin structure, act on the kappa-receptor. We have now cloned a human genomic DNA segment containing the preproenkephalin B gene. The structural organization of this gene resembles those of the genes encoding the other opioid peptide precursors, that is, preproenkephalin A and the corticotropin-beta-lipotropin precursor (ACTH-beta-LPH precursor). The primary structure of human preproenkephalin B has been deduced from the gene sequence. The amino acid sequence homology observed between preproenkephalin B and preproenkephalin A, together with the similarity between their gene organizations, suggests that the two genes have been generated from a common ancestor by gene duplication.

Cloning and sequence analysis of cDNA for porcine beta-neo-endorphin/dynorphin precursor.

The primary structure of a precursor protein that contains beta-neo-endorphin, dynorphin and a third leucine-enkephalin sequence with a carboxyl extension has been deduced from the nucleotide sequence of cloned DNA complementary to the porcine hypothalamic mRNA encoding it. The three peptides are each bounded by Lys-Arg. This precursor protein, like adrenal preproenkephalin and the corticotropin/beta-lipotropin precursor, comprises multiple repetitive units and a cysteine-containing amino-terminal sequence preceded by a signal peptide.

Isolation and characterization of the bovine corticotropin/beta-lipotropin precursor gene.

The entire bovine corticotropin/beta-lipotropin precursor gene has been isolated as a set of overlapping genomic DNA fragments which extend over a length of approximately 17000 base pairs. Restriction mapping of the cloned DNA fragments and nucleotide sequence analysis of the whole mRNA-coding segments and their surrounding regions have established that the corticotropin/beta-lipotropin precursor gene is approximately 7300-base-pairs long and contains two intervening sequences; one with an approximate length of 4000 base pairs is located within the segment encoding the 5'-untranslated region of the mRNA, and the other with an approximate length of 220 base pairs interrupts the protein-coding sequence near the signal peptide region. Sequence analysis of more than 200 base pairs preceding the proximal end of the corticotropin/beta-lipotropin precursor gene has revealed a 'Hogness box' and a variant of the model sequence d(G-G-TC-C-A-A-T-C-T) as well as palindrome structures as observed in other eukaryotic genes. Furthermore, some sequence similarities in the 5'-flanking region are found between the corticotropin/beta-lipotropin precursor gene and the mouse alpha-globin and beta-globin genes, all of which are negatively regulated by glucocorticoids. At least four homologous repetitive sequences are distributed at 3000-5000-base-pair distances in the corticotropin/beta-lipotropin precursor gene region; two such sequences are located in the 5'-flanking region, and one within each intervening sequence. Blot hybridization analysis of bovine pituitary nuclear RNA has indicated that the entire corticotropin/beta-lipotropin precursor gene is transcribed into a primary hnRNA product, which is then spliced to form the mature mRNA.

The protein-coding sequence of the bovine ACTH-beta-LPH precursor gene is split near the signal peptide region.

The pituitary hormones corticotropin (ACTH) and beta-lipotropin (beta-LPH) are formed from a large common precursor. Recently, we have elucidated the whole primary structure of the bovine ACTH-beta-LPH precursor (designated alternatively as preproopiocortin) by determining the nucleotide sequence of cloned DNA complementary to the mRNA coding for the precursor protein. The amino acid sequence assigned has disclosed a characteristic repetitive structure of the ACTH-beta-LPH precursor. The repetitive units of the precursor protein each contain a melanotropin (MSH) sequence (alpha-, beta- or gamma-MSH) as well as other peptide components such as beta-endorphin and corticotropin-like intermediate lobe peptide (CLIP). The repetitive units as well as their peptide components are each bounded by paired basic amino acid residues, which apparently represent the sites of proteolytic processing. Several studies have confirmed the translational initiation site and protein structure assigned (see also ref. 11 and refs therein). In view of the recent knowledge about the organization of eukaryotic genes (see refs 12, 13 for reviews), it would be of particular interest to investigate the relationship between the repetitive structure of the ACTH-beta-LPH precursor containing different functional components and the arrangement of the protein-coding sequence in its gene. We have now isolated and characterized bovine genomic DNA fragments encoding this precursor protein and have demonstrated that the protein sequence is encoded by two non-consecutive DNA segments. An intron (intervening sequence) of approximately 2.2 kilobase pairs separates the smaller exon (mRNA-coding sequence), which contains the gene sequence encoding the signal peptide, from the larger exon, which contains the gene sequence for most of the protein structure, including the known biologically active component peptides.