Plasma protein and supplemental isoleucine in milk replacers for dairy calves.

We measured the effects of milk replacers containing 0, 33, 66, or 100% of the total replaceable whey protein as bovine plasma protein (PP), without or with Ile supplementation, on the intake, growth, and health of 124 male Holstein calves for 35d. Milk replacers were formulated to contain 18% crude protein and 20% fat, with contents of Lys and Met equalized. When fed to calves at 1.5% of body weight (dry matter basis) under thermoneutral conditions, diets were predicted to allow average daily gains of 0.55kg/d based on metabolizable energy or 0.40kg/d based on apparent digestible protein. Protein supply was more limiting than energy so that differences in protein use could be detected. Dry matter intakes decreased with increased PP, irrespective of Ile supplementation. Final body weights decreased linearly with increasing PP, regardless of Ile supplementation. Average daily gain tended to be affected in a quadratic manner as PP increased, either with or without Ile supplementation; average daily gain and gain-feed ratio were greatest for calves fed diets containing 33% PP and lowest for calves fed 100% PP. The analyzed Lys content in the milk replacers was variable compared with formulated values, and this may have affected growth results. However, the gain-Lys ratio was affected by an interaction of the linear effect of increasing PP with Ile supplementation: it decreased with increasing PP but was improved by supplementation with Ile for calves fed 100% PP. Body measurements decreased with increasing PP inclusion; only decreased heart girth was reversed with Ile supplementation. The lowest and highest inclusion of PP, regardless of Ile supplementation, decreased the occurrence of scours compared with the control diet (all whey protein). Calves fed the lowest and highest PP without Ile supplementation also had fewer total days of scours in the first 21d. In addition, calves fed 100% PP without supplementation of Ile had fewer days of medication compared with the control diet. Even at the highest PP inclusion, average daily gain was minimally affected if Ile was supplemented. Growth rates, gain-feed ratio, and gain-Lys ratio were decreased at higher PP inclusion, but Ile overcame part of the reduction in gain-Lys ratio for 100% PP. Additional titration studies will have to be conducted to determine optimal PP inclusion rates, with a focus on supplementation of potentially limiting essential AA, as well as effects at higher growth rates.

Sulforaphane induces phase II detoxication enzymes in mouse skin and prevents mutagenesis induced by a mustard gas analog.

Mustard gas, used in chemical warfare since 1917, is a mutagenic and carcinogenic agent that produces severe dermal lesions for which there are no effective therapeutics; it is currently seen as a potential terrorist threat to civilian populations. Sulforaphane, found in cruciferous vegetables, is known to induce enzymes that detoxify compounds such as the sulfur mustards that react through electrophilic intermediates. Here, we observe that a single topical treatment with sulforaphane induces mouse epidermal levels of the regulatory subunit of glutamate-cysteine ligase, the rate-limiting enzyme in glutathione biosynthesis, and also increases epidermal levels of reduced glutathione. Furthermore, a glutathione S-transferase, GSTA4, is also induced in mouse skin by sulforaphane. In an in vivo model in which mice are given a single mutagenic application of the sulfur mustard analog 2-(chloroethyl) ethyl sulfide (CEES), we now show that therapeutic treatment with sulforaphane abolishes the CEES-induced increase in mutation frequency in the skin, measured four days after exposure. Sulforaphane, a natural product currently in clinical trials, shows promise as an effective therapeutic against mustard gas.

Specific mutations induced by triplex-forming oligonucleotides in mice.

Triplex-forming oligonucleotides (TFOs) recognize and bind to specific duplex DNA sequences and have been used extensively to modify gene function in cells. Although germ line mutations can be incorporated by means of embryonic stem cell technology, little progress has been made toward introducing mutations in somatic cells of living organisms. Here we demonstrate that TFOs can induce mutations at specific genomic sites in somatic cells of adult mice. Mutation detection was facilitated by the use of transgenic mice bearing chromosomal copies of the supF and cII reporter genes. Mice treated with a supF-targeted TFO displayed about fivefold greater mutation frequencies in the supF gene compared with mice treated with a scrambled sequence control oligomer. No mutagenesis was detected in the control gene (cII) with either oligonucleotide. These results demonstrate that site-specific, TFO-directed genome modification can be accomplished in intact animals.

Triplex-directed modification of genes and gene activity.

Oligonucleotides offer enormous potential for manipulating gene function in cells and, as such, constitute a promising new class of pharmaceutical agents. Oligonucleotides that form triple helices (triplexes) at specific DNA sequences in defined genes can be used to reduce transcription selectively, to introduce site-specific mutations or to stimulate gene-specific targeted recombination.

Chemotherapeutic characterization in mice of 2-amino-9-beta-D-ribofuranosylpurine-6-sulfinamide (sulfinosine), a novel purine nucleoside with unique antitumor properties.

In preclinical investigations performed in mice, 2-amino-9-beta-D-ribofuranosyl purine-6-sulfinamide (sulfinosine), a novel derivative of 6-thioguanosine (6TGR), was active against six solid tumors and four strains of experimental leukemia. Sulfinosine penetrated the central nervous system more readily than did 6TGR and, when given repeatedly, was much more effective in the treatment of L1210 leukemia, being curative for some mice. Other findings of major interest to us were the different dosing characteristics of sulfinosine and 6TGR, the divergent efficiencies of the two drugs in generating cellular resistance, and the activity of sulfinosine against experimental leukemias refractory to 6TGR and other experimental or clinically used chemotherapeutic agents. The chemotherapeutic characterization of sulfinosine that evolved from these studies suggests that this agent may have unique properties that deserve clinical consideration. Both the dosing characteristics of the drug and its pronounced activity against thiopurine-resistant experimental leukemia favor the possibility that sulfinosine could be used to advantage in the treatment of human leukemia unresponsive to 6-mercaptopurine or 6-thioguanine.

Oxidation of 2-amino-9-beta-D-ribofuranosylpurine-6-sulfenamide to the corresponding 6-sulfonamide facilitates changes in biologic characterization that include activity against thiopurine-refractory experimental leukemia.

Preclinical investigations in vivo revealed unexpected differences in the biological characteristics of 2-amino-9-beta-D-ribofuranosylpurine-6-sulfenamide (sulfenosine, 1) and 2-amino-9-beta-D-ribofuranosylpurine-6-sulfonamide (sulfonosine, 2), two novel but structurally related derivatives of 6-thioguanosine (6TGR). Strikingly, the addition of a fully oxidized sulfur atom at the 6 position of sulfenosine produced a purine derivative (sulfonosine) that was remarkably active against experimental leukemia resistant to treatment with either sulfenosine or 6TGR. This slight structural modification also appeared to influence solubility, scheduling capability, and oral activity as well as penetration of the central nervous system (CNS) and the onset of cellular resistance. These findings underscore the dramatic changes in biologic activity that can be produced by subtle modifications in molecular structure. We trust they may also contribute to the development of improved clinical therapy.

Triapine (3-aminopyridine-2-carboxaldehyde- thiosemicarbazone): A potent inhibitor of ribonucleotide reductase activity with broad spectrum antitumor activity.

Previous studies from our laboratories have shown that (a) Triapine() is a potent inhibitor of ribonucleotide reductase activity and (b) hydroxyurea-resistant L1210 leukemia cells are fully sensitive to Triapine. In an analogous manner, Triapine was similarly active against the wild-type and a hydroxyurea-resistant subline of the human KB nasopharyngeal carcinoma. Triapine was active in vivo against the L1210 leukemia over a broad range of dosages and was curative for some mice. This agent also caused pronounced inhibition of the growth of the murine M109 lung carcinoma and human A2780 ovarian carcinoma xenografts in mice. Optimum anticancer activity required twice daily dosing due to the duration of inhibition of DNA synthesis which lasted about 10 hr in L1210 cells treated with Triapine in vivo. DNA synthesis in normal mouse tissues (i.e. duodenum and bone marrow) uniformly recovered faster than that in L1210 leukemia cells, demonstrating a pharmacological basis for the therapeutic index of this agent. Triapine was more potent than hydroxyurea in inhibiting DNA synthesis in L1210 cells in vivo, and the effects of Triapine were more pronounced. In addition, the duration of the inhibition of DNA synthesis in leukemia cells from mice treated with Triapine was considerably longer than in those from animals treated with hydroxyurea. Combination of Triapine with various classes of agents that damage DNA (e.g. etoposide, cisplatin, doxorubicin, and 1-acetyl-1,2-bis(methylsulfonyl)-2-(2-chloroethyl)hydrazine) resulted in synergistic inhibition of the L1210 leukemia, producing long-term survivors of tumor-bearing mice treated with several dosage levels of the combinations, whereas no enhancement of survival was found when Triapine was combined with gemcitabine or cytosine arabinoside. The findings demonstrate the superiority of Triapine over hydroxyurea as an anticancer agent and further suggest that prevention by Triapine of repair of DNA lesions created by agents that damage DNA may result in efficacious drug combinations for the treatment of cancer.

Triplex targets in the human rhodopsin gene.

We have explored the application of triplex technology to the human rhodopsin gene, which encodes a G-protein-linked receptor involved in the genetic disorder autosomal dominant retinitis pigmentosa (ADRP). Our results support the hypothesis that most human genes contain high-affinity triplex sites and further refine the rules governing identification and successful targeting of triplex-forming oligonucleotides (TFOs) to these sites. Using a computer search for sites 15 nucleotides in length and greater than 80% purine, we found 143 distinct sites in the rhodopsin gene and comparable numbers of sites in several other human genes. By applying more stringent criteria, we selected 17 potential target sites in the rhodopsin gene, screened them with a plasmid binding assay, and found 8 that bound TFOs with submicromolar affinity (Kd = 10(-)9-10(-)7 M). We compared purine (GA) and mixed (GT) TFOs at each site, and found that GA-TFOs consistently bound with higher affinity, and were less sensitive to pyrimidine interruptions in the target strand. High G-content favored high-affinity binding; only sites with >54% G-content bound TFOs with Kd

High-affinity triple helix formation by synthetic oligonucleotides at a site within a selectable mammalian gene.

Specific recognition of duplex DNA by a single-stranded oligonucleotide via the formation of triplex DNA is a rational approach for targeting specific regions of a genome. By screening a number of potential target sites for triple helix formation within mammalian genes that allow genetic selection in cell culture, we have identified a site within intron 1 of the hamster adenine phosphoribosyltransferase (APRT) gene that specifically binds a triplex-forming oligodeoxyribonucleotide (TFO) with high affinity. Under optimal conditions for triplex formation, the equilibrium dissociation constant is in the nanomolar range (Kd = 7 x 10(-10) M). This high-affinity binding is very specific, as a 10(5)-fold excess of genomic DNA reduced triplex formation less than 10-fold, and within a 6928-bp plasmid bearing the APRT gene, only restriction fragments containing the intron 1 site were found to bind the TFO. Results of DNase I protection assays were consistent with the TFO binding in an antiparallel orientation via reverse Hoogsteen hydrogen bonds in the major groove of the duplex. We have examined the kinetics of triplex formation as well as the effects of ionic composition and chemical modifications of the TFO on triplex formation. While divalent cations were not required for triplex formation, Mg2+ stabilized the triplex apparently through inhibition of TFO dissociation, with a mean bound lifetime of > 17 h for the triplex at Mg2+ concentrations above 5 mM.(ABSTRACT TRUNCATED AT 250 WORDS)

High-efficiency triple-helix-mediated photo-cross-linking at a targeted site within a selectable mammalian gene.

Targeting damage to specific sites in the genome represents an attractive approach to manipulating gene function in mammalian cells. To test the applicability of triple-helix formation as a means for achieving precisely timed site-specific damage within a mammalian gene, a triplex-forming oligodeoxyribonucleotide (TFO) that binds with high affinity to a specific site within the hamster adenine phosphoribosyltransferase (APRT) gene was modified with the photochemically reactive psoralen derivative 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT). The modified TFO, psorTFO1, bound with high affinity to a target site within intron 1 of the APRT gene. Upon irradiation, photomonoadducts (i.e., covalent adducts of psorTFO1 to one strand of the target duplex) were formed with high efficiency (approximately 50%). Introduction of 5'-TpA sequences (the preferred site for psoralen-induced photo-cross-links) at or near the triplex junction leads to increased efficiency of total photoadduct formation and to efficient formation of products that had the electrophoretic mobility on denaturing PAGE expected for three-stranded photo-cross-links (i.e., products containing psorTFO1 covalently linked to both strands of the duplex). Their identities as cross-links were verified by (1) identical electrophoretic mobility of products formed with either duplex strand radiolabeled and (2) coprecipitation of the radiolabeled duplex strand with its complementary biotinylated strand following denaturation. In addition, the cross-links were completely reversible upon irradiation at 254 nm, as expected for psoralen-mediated cross-links. The yield and distribution of photoadducts depended on 5'-TpA position. The most efficient photoadduct formation (approximately 90%) and photo-cross-link formation (approximately 90% of total photoadducts) were observed for a 5'-TpA adjacent to the triplex junction, with significant, but lower, cross-linking efficiency within three base pairs of the junction. Molecular models of the psoralen-conjugated triplex with its six-carbon linker suggested a simple explanation for this distance dependence: facile intercalation near the triplex/duplex junction, with increasing strain required for intercalation at more distant sites. These results indicate that psorTFO1 allows for DNA damage with high precision and high efficiency, and the likely proportion of monoadducts and cross-links can be estimated from the target sequence.

Manipulating the mammalian genome by homologous recombination.

Gene targeting in mammalian cells has proven invaluable in biotechnology, in studies of gene structure and function, and in understanding chromosome dynamics. It also offers a potential tool for gene-therapeutic applications. Two limitations constrain the current technology: the low rate of homologous recombination in mammalian cells and the high rate of random (nontargeted) integration of the vector DNA. Here we consider possible ways to overcome these limitations within the framework of our present understanding of recombination mechanisms and machinery. Several studies suggest that transient alteration of the levels of recombination proteins, by overexpression or interference with expression, may be able to increase homologous recombination or decrease random integration, and we present a list of candidate genes. We consider potentially beneficial modifications to the vector DNA and discuss the effects of methods of DNA delivery on targeting efficiency. Finally, we present work showing that gene-specific DNA damage can stimulate local homologous recombination, and we discuss recent results with two general methodologies--chimeric nucleases and triplex-forming oligonucleotides--for stimulating recombination in cells.

Chromosome targeting at short polypurine sites by cationic triplex-forming oligonucleotides.

Triplex-forming oligonucleotides (TFOs) bind specifically to duplex DNA and provide a strategy for site-directed modification of genomic DNA. Recently we demonstrated TFO-mediated targeted gene knockout following systemic administration in animals. However, a limitation to this approach is the requirement for a polypurine tract (typically 15-30 base pairs (bp)) in the target DNA to afford high affinity third strand binding, thus restricting the number of sites available for effective targeting. To overcome this limitation, we have investigated the ability of chemically modified TFOs to target a short (10 bp) site in a chromosomal locus in mouse cells and induce site-specific mutations. We report that replacement of the phosphodiester backbone with cationic phosphoramidate linkages, either N,N-diethylethylenediamine or N,N-dimethylaminopropylamine, in a 10-nucleotide, psoralen-conjugated TFO confers substantial increases in binding affinity in vitro and is required to achieve targeted modification of a chromosomal reporter gene in mammalian cells. The triplex-directed, site-specific induction of mutagenesis in the chromosomal target was charge- and modification-dependent, with the activity of N,N-diethylethylenediamine > N,N-dimethylaminopropylamine phosphodiester, resulting in 10-, 6-, and <2-fold induction of target gene mutagenesis, respectively. Similarly, N,N-diethylethylenediamine and N,N-dimethylaminopropylamine TFOs were found to enhance targeting at a 16-bp G:C bp-rich target site in a chromatinized episomal target in monkey COS cells, although this longer site was also targetable by a phosphodiester TFO. These results indicate that replacement of phosphodiester bonds with positively charged N,N-diethylethylenediamine linkages enhances intracellular activity and allows targeting of relatively short polypurine sites, thereby substantially expanding the number of potential triplex target sites in the genome.

Psoralen photo-cross-linking by triplex-forming oligonucleotides at multiple sites in the human rhodopsin gene.

Targeting DNA damage by triplex-forming oligonucleotides (TFOs) represents a way of modifying gene expression and structure and a possible approach to gene therapy. We have determined that this approach can deliver damage with great specificity to sites in the human gene for the G-protein-linked receptor rhodopsin, mutations of which can lead to the genetic disorder autosomal dominant retinitis pigmentosa. We have introduced DNA monoadducts and interstrand cross-links at multiple target sites within the gene using TFOs with a photoactivatable psoralen group at the 5'-end. The extent of formation of photoadducts (i.e., monoadducts and cross-links) was measured at target sites with a 5'-ApT sequence at the triplex-duplex junction and at a target site with 5'-ApT and 5'-TpA sequences located four and seven nucleotides away, respectively. To improve psoralen reactivity at more distant sites, psoralen moieties were attached to TFOs with nucleotide "linkers" from two to nine nucleotides in length. High-affinity binding was maintained with linkers of up to 10 nucleotides, but affinities tended to decrease somewhat with increasing linker length due to faster dissociation kinetics. DNase I footprinting indicated little, if any, interaction between linkers and the duplex. Psoralen-TFO conjugates formed DNA cross-links with high efficiency (56-65%) at 5'-ApT sequences located at triplex junctions. At a 5'-ApT site four nucleotides away, the efficiency varied with linker length; a four-nucleotide linker gave the highest efficiency. Duplexes with 5'-TpA and 5'-ApT sites two nucleotides away, in otherwise identical sequences, were cross-linked with efficiencies of 56 and 38%, respectively. These results indicate that TFO-linker-psoralen conjugates allow simultaneous, efficient targeting of multiple sites in the human rhodopsin gene.

Chromosomal mutations induced by triplex-forming oligonucleotides in mammalian cells.

Specific recognition of a region of duplex DNA by triplex-forming oligonucleotides (TFOs) provides an attractive strategy for genetic manipulation. Based on this, we have investigated the ability of the triplex-directed approach to induce mutations at a chromosomal locus in living cells. A mouse fibroblast cell line was constructed containing multiple chromosomal copies of the lambdasupFG1 vector carrying the supFG1 mutation-reporter gene. Cells were treated with specific (psoAG30) or control (psoSCR30) psoralen-conjugated TFOs in the presence and absence of UVA irradiation. The results demonstrated a 6- to 10-fold induction of supFG1 mutations in the psoAG30-treated cells as compared with psoSCR30-treated or untreated control cells. Interestingly, UVA irradiation had no effect onthe mutation frequencies induced by the psoralen-conjugated TFOs, suggesting a triplex-mediated but photoproduct-independent process of mutagenesis. Sequencing data were consistent with this finding since the expected T.A-->A.T transversions at the predicted psoralen crosslinking site were not detected. However, insertions and deletions were detected within the triplex binding site, indicating a TFO-specific induction of mutagenesis. This result demonstrates the ability of triplex-forming oligonucleotides to influence mutation frequencies at a specific site in a mammalian chromosome.

Triplex-induced recombination in human cell-free extracts. Dependence on XPA and HsRad51.

Triple helix-forming oligonucleotides (TFOs) can bind to polypurine/polypyrimidine regions in DNA in a sequence-specific manner. Triple helix formation has been shown to stimulate recombination in mammalian cells in both episomal and chromosomal targets containing direct repeat sequences. Bifunctional oligonucleotides consisting of a recombination donor domain tethered to a TFO domain were found to mediate site-specific recombination in an intracellular SV40 vector target. To elucidate the mechanism of triplex-induced recombination, we have examined the ability of intermolecular triplexes to provoke recombination within plasmid substrates in human cell-free extracts. An assay for reversion of a point mutation in the supFG1 gene in the plasmid pSupFG1/G144C was established in which recombination in the extracts was detected upon transformation into indicator bacteria. A bifunctional oligonucleotide containing a 30-nucleotide TFO domain linked to a 40-nucleotide donor domain was found to mediate gene correction in vitro at a frequency of 46 x 10(-)5, at least 20-fold above background and over 4-fold greater than the donor segment alone. Physical linkage of the TFO to the donor was unnecessary, as co-mixture of separate TFO and donor segments also yielded elevated gene correction frequencies. When the recombination and repair proteins HsRad51 and XPA were depleted from the extracts using specific antibodies, the triplex-induced recombination was diminished, but was either partially or completely restored upon supplementation with the purified HsRad51 or XPA proteins, respectively. These results establish that triplex-induced, intermolecular recombination between plasmid targets and short fragments of homologous DNA can be detected in human cell extracts and that this process is dependent on both XPA and HsRad51.

In vivo stability and kinetics of absorption and disposition of 3' phosphopropyl amine oligonucleotides.

Development of oligonucleotide derivatives as therapeutic agents requires an understanding of their pharmacokinetic behavior. The in vivo disposition and stability of a prototype of such compounds are reported here. The compound studied, a relatively G-rich 38 base 3' phosphopropyl amine oligonucleotide (TFO-1), was cleared from the circulation with a half-life of approximately 10 minutes, displaying distribution kinetics consistent with a two compartment model. TFO-1 was also readily absorbed into circulation from the peritoneal cavity. All tissues examined except brain accumulated the compound reaching concentrations calculated to be in the micromolar range. TFO-1 was found to be stable in circulation and in tissues in that a large fraction of intact material was detected 8 hours after injection, as assessed by gel electrophoresis. Approximately 20-30% of the injected dose was excreted in the urine over an 8 hour period. These results suggest that G-rich oligonucleotides, minimally modified at the 3' end, are relatively stable in vivo and have distribution kinetics favorable to use as therapeutic agents.