Chemoselective high-throughput purification mediated by solid-supported reagents: its application to the first 6, 9-disubstituted purine library synthesis.

A new application of solid-supported reagents was developed to separate the alkylated N7/N9 regioisomers derived from commercially available 2-amino-6-chloropurine. Simple filtration through an alumina/H+ pad or scavenging by AG/Dowex-50W-X8 resin provides diverse N9 regioisomers selectively in moderate yields with high purities (>90%). This purification method can be conveniently used in a high-throughput format and facilitates the synthesis of a purine library without laborious regioisomer separation and aqueous work-up. The first library synthesis of 6,9-disubstituted purines is reported using the combination of this novel separation method in conjunction with polymer-supported reagents.

Antisense inhibition of the p65 subunit of NF-kappa B blocks tumorigenicity and causes tumor regression.

The NF-kappa B transcription factor, composed of two proteins, p50 and p65, is a pleiotropic activator that participates in the induction of a wide variety of cellular genes. Various cell adhesion molecules have NF-kappa B binding sites and may play an important role in inflammatory response, tumorigenicity, and metastasis. In an earlier study, we demonstrated that adhesion of diverse transformed cells was blocked by antisense inhibition of the p65 subunit of NF-kappa B. Since cell-substratum interactions play an important role in tumorigenicity, we reasoned that antisense p65 could inhibit tumorigenicity. In diverse transformed cell lines, phosphorothioate antisense oligonucleotides to p65 inhibited in vitro growth, reduced soft-agar colony formation, and eliminated the ability of cells to adhere to an extracellular matrix. Stable transfectants of a fibrosarcoma cell line expressing dexamethasone-inducible antisense RNA to p65 showed inhibition of in vitro growth and in vivo tumor development. In response to inducible expression of antisense RNA, a pronounced tumor regression was seen in nude mice. The administration of antisense but not sense p65 oligonucleotides caused a pronounced inhibition of tumorigenicity in nude mice injected with diverse tumor-derived cell lines. Inhibitors of NF-kappa B function may thus be useful in the treatment of cancer.

Cloning and expression of murine IL-12.

Human IL-12 (NK cell stimulatory factor, cytotoxic lymphocyte maturation factor) is a heterodimeric cytokine that can act as a growth factor for activated human T and NK cells, enhance the lytic activity of human NK/lymphokine-activated killer cells, and stimulate the production of IFN-gamma by resting human PBMC. Because in our hands, human IL-12 did not elicit similar responses in murine lymphocytes, we have cloned and expressed the murine IL-12 subunit cDNA in order to obtain recombinant protein for murine studies. Comparison of the predicted amino acid sequences of the murine subunits with their human counterparts revealed that the p40 subunits are more highly conserved than the p35 subunits (70% vs 60% identity, respectively). The sizes of the p35 and p40 subunit mRNA were estimated to be 1.5 kb and 2.6 kb, respectively. RNA blot analysis showed that p35 mRNA was expressed in lymphoid tissues (spleen, thymus) and nonlymphoid tissues (lung, brain), whereas p40 mRNA expression was only detected in lymphoid cells. Incubation of splenocytes with pokeweed mitogen did not significantly affect p35 mRNA levels, however, it resulted in a decrease of p40 mRNA. Coexpression of the murine p35 and p40 cDNA clones in COS cells resulted in the secretion of IL-12, which was active in human and mouse T cell proliferation, murine NK cell activation, and murine IFN-gamma induction assays. Transfection of each subunit cDNA alone did not result in measurable secreted IL-12 activity. A hybrid heterodimer consisting of murine p35 and human p40 subunits retained bioactivity on murine cells; however, the combination of human p35 and murine p40 was completely inactive on murine cells. These results indicate that the observed inability of human IL-12 to act on murine cells is largely determined by the p35 subunit.

Coexpression of two distinct genes is required to generate secreted bioactive cytotoxic lymphocyte maturation factor.

Cytotoxic lymphocyte maturation factor (CLMF) is a disulfide-bonded heterodimeric lymphokine that (i) acts as a growth factor for activated T cells independent of interleukin 2 and (ii) synergizes with suboptimal concentrations of interleukin 2 to induce lymphokine-activated killer cells. We now report the cloning and expression of both human CLMF subunit cDNAs from a lymphoblastoid B-cell line, NC-37. The two subunits represent two distinct and unrelated gene products whose mRNAs are coordinately induced upon activation of NC-37 cells. Coexpression of the two subunit cDNAs in COS cells is necessary for the secretion of biologically active CLMF; COS cells transfected with either subunit cDNA alone do not secrete bioactive CLMF. Recombinant CLMF expressed in mammalian cells displays biologic activities essentially identical to natural CLMF, and its activities can be neutralized by monoclonal antibodies prepared against natural CLMF. Since this heterodimeric protein displays the properties of an interleukin, we propose that CLMF be given the designation interleukin 12.

DNA methylation: sequences flanking C-G pairs modulate the specificity of the human DNA methylase.

Synthetic single-stranded oligodeoxynucleotides of known sequence have been used as in vitro substrates for a partially purified HeLa cell DNA methylase. Although most oligonucleotides tested cannot be used by the HeLa DNA methylase in vitro, we have found a unique 27mer, containing 2 C-G pairs, that is an excellent substrate for the enzyme. Analysis of the methylation of the 27mer, its derivatives and other oligomer substrates reveal that the HeLa DNA methylase does not significantly methylate an oligomer which contains just one C-G pair. In addition, only one of the two C-G pairs in the 27mer is methylated and this methylation is abolished if the other C-G pair is converted to a C-A pair. Furthermore, the HeLa enzyme apparently cannot methylate C-G pairs located in compounds containing a high A + T content. The most efficient methylation occurs with multiple separated C-G pairs in a compound with a high G + C content (greater than 65%). The results suggest that clustering of C-G pairs in regions of the DNA high in G + C content may be the preferred site for DNA methylation in vivo.

Synthesis and pharmacological evaluation of a series of analogues of 1-methylisoguanosine.

A series of analogues of the pharmacologically active marine natural product 1-methylisoguanosine (1) was evaluated for biological activity in muscle relaxant, cardiovascular, antiinflammatory, and antiallergic tests. Modifications at the 1 position produced the ethyl, n-butyl, n-octyl, and phenyl derivatives 3-6, respectively. Substitutions at the 8 position provided the bromo, hydrazino and amino compounds 9-11. Modification at the 5' position yielded the deoxy, iodo, and phosphate derivatives 15, 13, and 16, as well as the cyclic 3',5'-phosphate 17. The synthesis of the C-nucleoside analogue 19 was achieved from the beta-D-ribofuranosylcarboximidic ester 20. The acyclic analogue 29 and the beta-D-arabinofuranosyl derivative 35 were both synthesized by reaction of methyl isocyanate with the appropriately protected aminocyanoimidazole precursors 28 and 32. 1-Methylxanthosine (12), isoguanosine (7), and 2-methoxyadenosine (18) were also synthesized. At doses up to 100 mg/kg po, the 5'-phosphate 16, cyclic 3',5'-phosphate 17, and the O-methylated analogue 2-methoxyadenosine 18 were active in producing muscle relaxation and hypothermia. These compounds possessed antiallergic activity and produced dose-dependent falls in mean blood pressure and heart rate as did the 1-ethyl (3) and 1-n-butyl (4) analogues. In general, antiinflammatory activity paralleled the other results, except that the cyclic 3',5'-phosphate 17 was inactive at the dose tested, while the 3,5'-anhydronucleoside 14 was weakly active and displayed antiallergic effects.

Synthesis of arabinofuranosyl derivatives of 3-deazaguanine.

Synthesis of four arabinofuranosyl derivatives of the antitumor agent 3-deazaguanine is described. By the use of 13C and 1H nuclear magnetic resonance spectroscopy, the structures of these nucleosides were established to be alpha and beta pairs of N-7 and N-9 arabinosides of 3-deazaguanine. In contrast to 3-deazaguanine and its ribosyl derivative, the nucleosides described in this paper were found to be inactive against Sarcoma 180 in mice at 100 mg/kg.

Chemical synthesis of two deoxyribododecanucleotides for the attachment of restriction termini to an artificial minigene.

In order to permit in vivo cloning of an artificial minigene designed to code for a modified S-peptide, the phosphodiester method for the chemical synthesis of two dodecadeoxyribonucleotides is described. Each of the latter possesses antiparallel complementarity to one of the two minigene strands and to the single-stranded EcoRI-generated end. They can thus serve as cohesive termini ("splints") for polynucleotide ligase joining.