The rTS gene codes for a naturally occurring antisense RNA to thymidylate synthase (TS) mRNA and two proteins (rTSalpha and rTSbeta). The role of the major protein product of rTS, rTSbeta has been linked to alterations in TS protein expression, but the precise function of rTSbeta is unknown. In this report we demonstrate that increased expression of rTSbeta is associated with the decrease in TS protein expression due to production of novel, diffusible signal molecules. These signal molecules are produced more abundantly when rTSbeta amounts are elevated. This hypothesis is supported by the demonstration that the rTSbeta-overproducing cell line H630-1 can downregulate TS protein in other cells without direct cellular contact. These cells are shown to secrete significant amounts of lipophilic metabolites derived from methionine, in contrast to cells that do not overproduce rTSbeta. In support of the hypothesis that rTSbeta is essential for the generation of these compounds, we demonstrate that rTSbeta can catalyze the transfer of the carboxyl carbon of methionine from S-adenosylmethionine to a lipophilic acceptor molecule in vitro. We propose rTS is involved in regulation of TS through a novel methionine-based signaling pathway.

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The rTSB protein has been hypothesized to synthesize signaling molecules that can down-regulate thymidylate synthase. These molecules share biological and chemical properties with acyl-homoserine lactones (AHL), suggesting some AHLs might act as rTS signaling mimics and downregulate thymidylate synthase. We have determined that the AHL, 3-oxododecanoyl homoserine lactone (3-oxo-C12-(L)-HSL) can down-regulate thymidylate synthase protein at 10 Mmol/L and reduce H630 (human colorectal cancer) growth by 50% at 23 Mmol/L (IC 50 ) in cell culture. At its IC 50 concentration, 3-oxo-C12-(L)-HSL reduces the apparent IC 50 of 5-fluorouracil (5-FU) from 1 Mmol/L to 80 nmol/L (12-fold) in a colony formation assay. 3-Oxo-C12-(L)-HSL enhances the activity of 5-fluorodeoxyuridine, tomudex, and taxol but not the activity of 5-fluorouridine, methotrexate or Adriamycin. The unexpected interaction with taxol probably results from effects of the AHL on tubulin expression. Differences in taxol sensitivity, tubulin, and cellular morphology between H630 and the thymidylate synthase and rTSB-overproducing, 5-FUresistant H630-1 cell line as determined by colony formation assays, Western analysis of one-dimensional and two-dimensional gels, and photomicroscopy confirm that cytoskeletal changes are induced by the AHL or by rTS signaling. Isozyme differences in thymidylate synthase and rTSB also exist in the two cell lines. Phosphorylation of rTSB amino acid S121 is shown to occur and is decreased at least 10-fold in the drug-resistant cells. The data presented provide support for further investigations of rTS signaling mimics as enhancers to thymidylate synthase-directed chemotherapy, evidence that the phosphorylation state of rTSB may be a marker for 5-FU resistance and a previously unrealized relationship between rTS signaling and the cytoskeleton. (Cancer Res 2005; 65(13): 5917-24)

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Synthesis of the antileukemic agent 4,4'-diacetyl-N,N-diphenylurea bis (guanylhydrazone) (DDUG) is described in detail. The compound was characterized in terms of its and 13C NMR spectra. To determine the importance of structural features to its biological activity, a series of related compounds was synthesized. The aminoguanidine groups in DDUG were replaced with other groups, such as thiosemicarbazone or nitroaminoguanidine. In addition, one or both H atoms of the urea moiety of DDUG were replaced with a methyl group. One urea N was replaced with 0, giving a urethane moiety. The two guanylhydrazone groups in DDUG were separated with one or two methylene groups, and the effects of such separation were investigated in relation to the biological activity of the compound. In contrast to the modifications that we carried out earlier in the phenyl ring, modifications at and around the urea moiety of DDUG caused decreases in the growth-inhibitory activity of the compound against mouse mammary adenocarcinoma (TA3) cells in culture. Preliminary results indicate that at least some antileukemic activity is retained on single N-methylation and on extension of the molecule with one methylene group. Most of the DDUG analogues inhibited DNA polymerase, and the degree of inhibition was less sensitive to structural modification in this system than in cellular test systems or in vivo.