Novel lysine-spermine conjugate inhibits polyamine transport and inhibits cell growth when given with DFMO.

Polyamines are ubiquitous molecules with multiple intracellular functions. Cells tightly regulate their levels through feedback mechanisms affecting synthesis, intracellular conversion, and transport. Because polyamines have an important role in regulating cell growth, they are a target for cancer therapeutic development. However, to effectively inhibit cell growth through polyamine depletion one needs to inhibit both polyamine synthesis and import. Although the mammalian polyamine transporter has not been cloned, we have identified ORI 1202, an N(1)-spermine-L-lysinyl amide, as an effective polyamine transport inhibitor. ORI 1202 prevents the cellular accumulation of [(3)H]spermidine over a 20-h test period. ORI 1202 (30-100 microM) effectively inhibits cell growth when used in conjunction with the polyamine synthesis inhibitor alpha-difluoromethylornithine (DFMO; > or =230 microM). Human breast, prostate, and bladder carcinoma cell lines and melanoma cell lines show ORI 1202 EC(50) values in the low micromolar range when tested in conjunction with DFMO. This cytostatic effect correlates with a reduction in the intracellular levels of putrescine and spermidine. When ORI 1202 (45 mg/kg, i.p., tidx5) and DFMO (1% in drinking water) were delivered over 14 days, MDA-MB-231 breast tumor xenografts in nude mice showed 50% growth inhibition. Polyamine depletion therapy provides a cytostatic therapy that could be useful against cancer and other diseases resulting from uncontrolled cell growth.

The Saccharomyces cerevisiae Prp5 protein has RNA-dependent ATPase activity with specificity for U2 small nuclear RNA.

The Saccharomyces cerevisiae protein Prp5 is a member of the "DEAD box" family of putative RNA-dependent ATPases and helicases. The protein was purified from Escherichia coli and determined to be an RNA-dependent ATPase. The ATPase activity is 7-fold more specific for full-length U2 than for any of the other small nuclear RNAs or nonspecific RNAs tested. An RNaseH assay in extracts was used to demonstrate that Prp5 mediates an ATP-dependent conformational change in the intact U2 small nuclear ribonucleoprotein. We propose that this conformational change makes the branch point pairing sequence of U2 RNA accessible for pairing with the intron allowing formation of the pre-spliceosome.

In vitro studies of the Prp9.Prp11.Prp21 complex indicate a pathway for U2 small nuclear ribonucleoprotein activation.

Pre-mRNA splicing takes place on a large ribonucleoprotein particle, the spliceosome which contains the five small nuclear ribonucleoproteins (snRNPs), U1, U2, U4, U5, and U6. In Saccharomyces cerevisiae the mRNA splicing factors, Prp9, Prp11, and Prp21, are necessary for addition of the U2 snRNP to the pre-mRNA in an early step of spliceosome assembly. This paper describes a study of interactions between these proteins and their role in spliceosome assembly. The proteins were expressed in Escherichia coli. Prp9 and Prp11 were purified by metal affinity chromatography. Prp21 was purified using a solubilization/renaturation protocol. We have combined these separately purified proteins and present direct evidence of a Prp9.Prp11.Prp21 protein complex that is functional in in vitro splicing assays. Characteristics of this Prp9.Prp11.Prp21 complex were further investigated using proteins synthesized in vitro. In addition, we found that Prp9, Prp11, and Prp21 influence the structure of the U2 snRNP in a manner that alters the accessibility of the branch point pairing region of the U2 snRNA to oligonucleotide- directed RNaseH cleavage. We present a model, based on the data presented here and in the accompanying paper, for a combined role of Prp9, Prp11, Prp21, and Prp5 in activating the U2 snRNP for assembly into the pre-spliceosome.

18S rRNA processing requires the RNA helicase-like protein Rrp3.

We report the identification of a new gene, RRP3 (rRNA processing), which is required for pre-rRNA processing. Rrp3 is a 60.9 kDa protein that is required for maturation of the 35S primary transcript of pre-rRNA and is required for cleavages leading to mature 18S RNA. RRP3 was identified in a PCR screen for DEAD box genes. DEAD box genes are part of a large family of proteins homologous to the eukaryotic transcription factor elF-4a. Most of these proteins are RNA-dependent ATPases and some of them have RNA helicase activity. This is the third yeast DEAD box protein that has been shown to be involved in rRNA assembly, but the only one required for the processing of 18S RNA. Mutants of the two other putative helicases, Spb4 and Drsl, both show processing defects in 25S rRNA maturation. In strains where Rrp3 is depleted, 35S precursor RNA is improperly processed. Cleavage normally occurs at sites A0O, Al and A2, but in the Rrp3 depletion stain cleavage occurs between A2 and B1. Rrp3 has been purified to homogeneity and has a weak RNA-dependent ATPase activity which is not specific for rRNA.

PCNA-induced DNA synthesis past cis-syn and trans-syn-I thymine dimers by calf thymus DNA polymerase delta in vitro.

Calf thymus proliferating cell nuclear antigen (PCNA) promoted DNA synthesis past cis-syn and trans-syn-I cyclobutane thymine dimers by calf thymus DNA polymerase delta (Pol delta) in vitro. Templates containing site-specific cis-syn and trans-syn-I thymine dimers were prepared via a combination of solid phase synthesis with photoproduct building blocks and DNA ligation. Extension of a 15-mer primer on the UV dimer-containing templates by Pol delta produced termination and bypass products in a dNTP and PCNA dependent manner. In the absence of PCNA and at dNTP concentrations varying between 1 and 100 microM, Pol delta could not bypass the cis-syn dimer and terminated elongation one nucleotide prior to the 3'-T of the dimer. DNA synthesis past the trans-syn-I dimer was even less efficient. In the presence of PCNA, termination occurred primarily one nucleotide prior to the 3'-T of both dimers at 1 microM dNTPs but opposite the 5'-T of the dimers at 100 microM dNTPs. In addition, under the latter conditions, bypass of the dimers was observed, to the extent of about 30% of the products for the cis-syn dimer and about 15% for the trans-syn-I dimer.

cis-syn thymine dimers are not absolute blocks to replication by DNA polymerase I of Escherichia coli in vitro.

Both Escherichia coli DNA polymerase I (pol I) and the large fragment of pol I (Klenow) were found to bypass a site-specific cis-syn thymine dimer, in vitro, under standard conditions. A template was constructed by ligating d(pCGTAT[c,s]TATGC), synthesized via a cis-syn thymine dimer phosphoramidite building block, to a 12-mer and 19-mer. The site and integrity of the dimer were verified by use of T4 denV endonuclease V. Extension of a 15-mer on the dimer-containing template by either pol I or Klenow led to dNTP and polymerase concentration dependent formation of termination and bypass products. At approximately 0.15 unit/microL and 1-10 microM in each dNTP, termination one prior to the 3'-T of the dimer predominated. At 100 microM in each dNTP termination opposite the 3'-T of the dimer predominated and bypass occurred. Bypass at 100 microM in each dNTP depended on polymerase concentration, reaching a maximum of 20% in 1 h at approximately 0.2 unit/microL, underscoring the importance of polymerase binding affinity for damaged primer-templates on bypass. Seven percent bypass in 1 h occurred under conditions of 100:10 microM dATP:dNTP bias, 1% under dTTP bias, and an undetectable amount under either dGTP or dCTP bias. At 100 microM in each dNTP, the ratio of pdA:pdG:pdC:pdT terminating opposite the 3'-T of the dimer was estimated to be 37:25:10:28. Sequencing of the bypass product produced under these conditions demonstrated that greater than 95% pdA was incorporated opposite both Ts of the dimer and that little or no frame shifting took place.(ABSTRACT TRUNCATED AT 250 WORDS)