Maintenance of telomeres is implicated in chromosome stabilization and cell immortalization. Telomerase, which catalyzes de novo synthesis of telomeres, is activated in germ cells and most cancers. Telomerase activity is regulated by gene expression for its catalytic subunit, TERT, whereas several lines of evidence have suggested a post‐translational regulation of telomerase activity. Here we identify the 14‐3‐3 signaling proteins as human TERT (hTERT)‐binding partners. A dominant‐negative 14‐3‐3 redistributed hTERT, which was normally predominant in the nucleus, into the cytoplasm. Consistent with this observation, hTERT‐3A, a mutant that could not bind 14‐3‐3, was localized into the cytoplasm. Leptomycin B, an inhibitor of CRM1/exportin 1‐mediated nuclear export, or disruption of a nuclear export signal (NES)‐like motif located just upstream of the 14‐3‐3 binding site in hTERT impaired the cytoplasmic localization of hTERT. Compared with wild‐type hTERT, hTERT‐3A increased its association with CRM1. 14‐3‐3 binding was not required for telomerase activity either in vitro or in cell extracts. These observations suggest that 14‐3‐3 enhances nuclear localization of TERT by inhibiting the CRM1 binding to the TERT NES‐like motif.

The mechanism of resistance to 5‐fluorouracil (5‐FU) was studied with NUGC‐3/5FU/L, a human stomach cancer cell line which had acquired resistance as a consequence of repeated 5‐day exposures to stepwise‐increasing concentrations of 5‐FU in vitro. NUGC‐3/5FU/L was 200‐fold and over 16‐fold resistant to 96‐h and 1‐h exposures to 5‐FU, respectively. NUGC‐3/5FU/L incorporated less 5‐FU into RNA, indicating resistance to the RNA‐directed action of 5‐FU. On the other hand, NUGC‐3/5FU/L also showed resistance to in situ thymidylate synthase (TS) inhibition by 5‐FU. Polymerase chain reaction‐single‐strand conformation polymorphism analysis of TS cDNA and a FdUMP ligand binding assay showed that quantitative and qualitative alterations of TS are not responsible for this resistance. In contrast, the ability to metabolize 5‐FU to its active metabolites, FUTP and FdUMP, was reduced in NUGC‐3/5FU/L. We found that not only the activities of uridine phosphorylase/kinase and orotate phosphoribosyl‐transferase (OPRT), but also the level of phosphoribosyl pyrophosphate, a cosubstrate for OPRT, were significantly lower in NUGC‐3/5FU/L than in the parent NUGC‐3. These results indicated that resistance to 5‐FU in NUGC‐3/5FU/L is due to reduced activities of 5‐FU‐anabolizing enzymes, but not to an alteration of TS. 2′‐Deoxyinosine effectively enhanced TS inhibition by 5‐FU in the resistant cells, thus markedly sensitizing them to 5‐FU.

A human stomach cancer cell line with acquired resistance to 5‐fluorouracil (5‐FU), NUGC‐3/5FU/L, has been found to possess reduced ability to convert 5‐FU into active metabolites. We attempted in vitro gene therapy for this 5‐FU‐resistant cell line. NUGC‐3 and NUGC‐3/5FU/L cells were infected with recombinant adenovirus (Ad) containing Escherichia coli uracil phosphoribosyltransferase (UPRT) gene driven by CAG promoter (CA), AdCA‐UPRT, and changes in their 5‐FU metabolism and sensitivity were investigated. Activities of orotate phosphoribosyltransferase increased from 10.2 and 1.56 (nmol/mg protein/30 min) in the uninfected cells of NUGC‐3 and NUGC‐3/5FU/L to 216 and 237, respectively, after the transfection of UPRT gene. The 5‐FU nucleotide level in the acid‐insoluble fraction increased from 7.32 to 15.9 (pmol/mg protein) in NUGC‐3 cells on infection with AdCA‐UPRT, and in NUGC‐3/5FU/L cells it increased from 1.91 to 21.4. The 50% growth‐inhibition concentration (IC50) was 12.7 μmol/liter for NUGC‐3 and much higher than 100 μmol/liter for NUGC‐3/5FU/L, indicating over 8‐fold resistance. NUGC‐3/5FU/L transfected with the UPRT gene showed very high sensitivity to 5‐FU with an IC50 of 3.2 μmol/liter. The high resistance in this metabolic activation‐deficient cell line was thus completely reversed by transduction of an exogenous gene coding for a 5‐FU‐anabolizing enzyme.

Key words: HT-29 human colon cancer -5-Fluorouracil-resistance -Dual actions of 5-FUExposure time 5-FU is a unique anticancer agent with two major pharmaco-biochemical actions: of its active metabolites, FUTP is fraudulently incorporated into RNA and impairs the multiple functions of RNA, while FdUMP blocks the catalytic activity of TS by forming a ternary covalent complex with its co-substrate, 5, 10-CH 2 -FH 4 , which inhibits DNA synthesis. Thus 5-FU potentially exerts both RNAand DNA-directed actions against target cells.One of the factors determining which action 5-FU preferentially exerts on the target cells seems to be their innate biochemical characteristics of 5-FU metabolism. If the resultant intracellular concentration of FUTP or FdUMP is lower than the effective level, 5-FU may exert only one of its dual actions. However, it seems to exert dual actions against the majority of cancer cells, although the efficacy of such actions varies. Another determinant is the length of the exposure time to 5-FU. We have previously developed a method for the kinetic analysis of the cell-killing action of anticancer agents which can classify them into cell cycle phase-nonspecific (type I) drugs and cell cycle phase-specific (type II) drugs. 1,2) According to this analysis, FUrd and FdUrd showed typical kinetic profiles for type I and II drugs, respectively. 5-FU had a FUrd-like kinetic profile for cell killing action when cells were exposed for a short time and a FdUrd-like profile when they were continuously exposed for a long time. 3, 4)These results suggested that 5-FU exerts RNA-directed action under short exposure conditions and DNA-directed action under long, continuous exposure conditions. The former requires a high 5-FU concentration, while for the latter, a low concentration is sufficient.In view of the dual actions of 5-FU, which depend on the exposure time, it might be possible to develop a subline which acquires resistance to short/long exposure to 5-FU, but does not show cross-resistance to long/short exposure. Based on this idea, we have established some 5-FU-resistant sublines of human gastro-intestinal cancer cell lines by repeated long-term exposure to 5-FU. However, we failed to obtain a subline of the type we had expected. In the present study, we tried repeated 1-h exposure of human colon cancer HT-29 cells to 5-FU, and established a subline named HT-29/5-FU/S. This subline is resistant to short-term exposure to 5-FU, exhibited no cross-resistance to long-term exposure to 5-FU, and had rather higher sensitivity than its parent cells.

No abstract