The complete 5284-nucleotide sequence ofthe double-stranded RNA genome of Leishmania RNA virus 1 (LRV1) was determined and contains three open reading frames (ORFs) on the plus (+) (mRNA) (3, 7). The 5.3-kilobase (kb) LRV1 dsRNA was purified by electrophoresis in 0.6% agarose/ TBE (0.09 M Tris borate/2 mM EDTA, pH 8.0) gels, electroelution, and ethanol precipitation. LRV1 Cloning. LRV1 cDNA clones were prepared from gel-purified RNA (5) or by PCR amplification. Their location within the LRV1 genome is shown in Fig. 1. First-strand cDNA was synthesized using mixed hexamer primers (LP series), oligo(dT) priming of Escherichia coli poly(A) polymerase-treated LRV1 RNA (U1 series); and oligonucleotides 87-19 and 87-20 (see Fig. 2) (LW or WAL series). Second strand was synthesized by the RNase H method (9), repaired, methylated, tinkered, and ligated into BamHI-digested pBS+ (LP series), EcoRI-digested AZAP (LJ series) or EcoRIdigested AZAPII (LW and WAL series) (10). Clones of 5' ends were obtained by PCR amplification of C-tailed (terminal deoxynucleotidyltransferase) first-strand cDNA synthesized\ using primers 90-122 and 87-19 for the (+)-and (-)-strand, respectively. Amplification with Bam-dG10 (CCGGATCCGGGGGGGGGG) and 90-145 or 89-195, respectively, used Replinase (DuPont/NEN) for 30 cycles of denaturation at 940C for 1 min, annealing at 450C for 1 mi, and extension at 720C for 2 min. The products were digested with BamHI and EcoRI (1P145 series), BamHI and Nhe I (1PN4 series), or BamHI and HindIll (1P195 series) and ligated into pBluescript II SK(-) (Stratagene) digested with the appropriate enzymes. For cloning the 3' ends, gel-purified dsRNA was C-tailed using poly(A) polymerase (BRL), cDNA was synthesized using Bam-dG10, and PCR amplification was carried out using 90-122 and 90-146 were used for first-strand cDNA synthesis and PCR amplification ofgel-purified LRV1 dsRNA for some internal LRV1 clones. Products were digested with EcoRI and ligated into EcoRI-digested pBluescript II SK(-) (PCR146-1 and P2R series).Sequence Analysis. Plasmid and PCR product DNA was sequenced by the dideoxy chain-termination method using Sequenase (United States Biochemical) (11) and sequence analysis was carried out using DNASTAR and PCFOLD (12) and CLUSTAL (13) software. Homology searches ofthe Swiss-Prot

Cancer cells can overcome the ability of polyamine biosynthesis inhibitors from completely depleting their internal polyamines by the importation polyamines from external sources. We have developed a group of lipophilic polyamine analogs that potently inhibit the cellular polyamine uptake system and greatly increase the effectiveness of polyamine depletion when used in combination with DFMO, a well-studied polyamine biosynthesis inhibitor. By the attachment of an length-optimized C 16 lipophilic substituent to the epsilon-nitrogen atom of our earlier lead compound, D-Lys-Spm (5), we have produced an analog, D-Lys(C 16 acyl)-Spm (11) with several orders of magnitude more potent cell growth inhibition on a variety of cultured cancer cell types including breast (MDA-MB-231), prostate (PC-3), melanoma (A375) and ovarian (SK-OV-3), among others. We discuss these results in the context of a possible membrane-catalyzed interaction with the extracellular polyamine transport apparatus. The resulting novel two-drug combination therapy targeting cellular polyamine metabolism has shown exceptional efficacy against cutaneous squamous cell carcinomas (SCC) in a transgenic ornithine decarboxylase (ODC) mouse model of skin cancer. A majority (88%) of large, aggressive SCCs exhibited complete or near-complete remission to this combination therapy, while responses to each agent alone were poor. The availability of a potent polyamine transport inhibitor allows, for the first time, for a real test of the hypothesis that starving cells of polyamines will lead to objective clinical response.

Using a recently developed autochthonous mouse model of squamous cell carcinoma (SCC), a combination therapy targeting polyamine metabolism was evaluated. The therapy combined 2-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (ODC), and MQT 1426, a polyamine transport inhibitor. In 1 trial lasting 4 weeks, combination therapy with 0.5% DFMO (orally, in the drinking water) and MQT 1426 (50 mg/kg i.p., bid) was significantly more effective than with either single agent alone when complete tumor response was the endpoint. In the combination group, 72% of SCCs responded completely vs. 21 and 0% for DFMO and MQT 1426, respectively. A second trial involved a 4-week treatment period followed by 6 weeks off-treatment. With apparent cures as an endpoint, combination therapy was again more effective than either agent alone: a 50% apparent cure rate was observed in the combination group vs. 7.7% in the DFMO group. MQT 1426 had no inhibitory effect on SCC ODC activity nor did it enhance the inhibition by DFMO, but SCC polyamine levels declined more rapidly when treated with combination therapy vs. DFMO alone. The apoptotic index in SCCs was transiently increased by combination therapy but not by DFMO alone. Thus, targeting both polyamine biosynthesis and polyamine transport from the tumor microenvironment enhances the efficacy of polyamine-based therapy in this mouse model of SCC. ' 2005 Wiley-Liss, Inc.

No abstract

In this paper we describe the synthesis and characterization of a series of simple spermine/amino acid conjugates, some of which potently inhibit the uptake of spermidine into MDA-MB-231 breast cancer cells. The presence of an amide in the functionalized polyamine appeared to add to the affinity for the polyamine transporter. The extensive biological characterization of an especially potent analogue from this series, the Lys-Spm conjugate (31), showed this molecule will be an extremely useful tool for use in polyamine research. It was shown that the use of 31 in combination with DFMO led to a cytostatic growth inhibition of a variety of cancer cells, even when used in the presence of an extracellular source of transportable spermidine. It was furthermore shown that this combination effectively reduced the cellular levels of putrescine and spermidine while not affecting the levels of spermine. These facts together with the nontoxic nature of 31 make it a novel lead for further anticancer development.