Characterization of a thymidylate synthase (TS)-inducible cell line: a model system for studying sensitivity to TS- and non-TS-targeted chemotherapies

Thymidylate synthase (TS) is responsible for the de novo synthesis of thymidylate, which is required for DNA synthesis and repair and which is an important target for fluoropyrimidines such as 5-fluorouracil (5-FU), and antifolates such as Tomudex (TDX), ZD9331, and multitargeted antifolate (MTA). To study the importance of TS expression in determining resistance to these agents, we have developed an MDA435 breast cancer-derived cell line with tetracycline-regulated expression of TS termed MTS-5. We have demonstrated that inducible expression of TS increased the IC(50) dose of the TS-targeted therapeutic agents 5-FU, TDX, and ZD9331 by 2-, 9- and 24-fold respectively. An IC(50) dose for MTA was unobtainable when TS was overexpressed in these cells, which indicated that MTA toxicity is highly sensitive to increased TS expression levels. The growth inhibitory effects of the chemotherapeutic agents CPT-11, cisplatin, oxaliplatin, and Taxol were unaffected by TS up-regulation. Cell cycle analyses revealed that IC(50) doses of 5-FU, TDX and MTA caused an S-phase arrest in cells that did not overexpress TS, and this arrest was overcome when TS was up-regulated. Furthermore, the S-phase arrest was accompanied by 2- to 4-fold increased expression of the cell cycle regulatory genes cyclin E, cyclin A, and cyclin dependent kinase 2 (cdk2). These results indicate that acute increases in TS expression levels play a key role in determining cellular sensitivity to TS-directed chemotherapeutic drugs by modulating the degree of S-phase arrest caused by these agents. Moreover, CPT-11, cisplatin, oxaliplatin, and Taxol remain highly cytotoxic in cells that overexpress TS.

E-Commerce Innovations in the Personal Tax System of the United Kingdom

This study finds evidence that attempts to reduce costs and error rates in the Inland Revenue through the use of e-commerce technology are flawed. While it is technically possible to write software that will record tax data, and then transmit it to the Inland Revenue, there is little demand for this service. The key finding is that the tax system is so complex that many people are unable to complete their own tax returns. This complexity cannot be overcome by well-designed software. The recommendation is to encourage the use of agents to assist taxpayers or simplify the tax system. The Inland Revenue is interested in saving administrative costs and errors by encouraging electronic submission of tax returns. To achieve these objectives, given the raw data it would seem clear that the focus should be on facilitating the work of agents.

Targeted cytoplasmic irradiation induces bystander responses.

The observation of radiation-induced bystander responses, in which cells respond to their neighbors being irradiated, has important implications for understanding mechanisms of radiation action particularly after low-dose exposure. Much of this questions the current dogma of direct DNA damage driving response in irradiated systems. In this study, we have used a charged-particle microbeam to target individual helium ions ((3)He(2+)) to individual cells within a population of radioresistant glioma cells cultured alone or in coculture with primary human fibroblasts. We found that even when a single cell within the glioma population was precisely traversed through its cytoplasm with one (3)He(2+) ion, bystander responses were induced in the neighboring nonirradiated glioma or fibroblasts so that the yield of micronuclei was increased by 36% for the glioma population and 78% for the bystander fibroblast population. Importantly, the yield of bystander-induced micronuclei was independent of whether the cytoplasm or nucleus of a cell was targeted. The bystander responses were fully eliminated when the populations were treated with 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide or filipin, which scavenge nitric oxide (NO) and disrupt membrane rafts, respectively. By using the probe 4-amino-5-methylamino-2',7'-difluorofluorescein, it was found that the NO level in the glioma population was increased by 15% after 1 or 10 cytoplasmic traversals, and this NO production was inhibited by filipin. This finding shows that direct DNA damage is not required for switching on of important cell-signaling mechanisms after low-dose irradiation and that, under these conditions, the whole cell should be considered a sensor of radiation exposure.

Nitric Oxide-Mediated Signaling in the Bystander Response of Individually Targeted Glioma Cells.

Bystander responses have been reported to be a major determinant of the response of cells to radiation exposure at low doses, including those of relevance to therapy. In this study, human glioblastoma T98G cell nuclei were individually irradiated with an exact number of helium ions using a single-cell microbeam. It was found that when only 1 cell in a population of approximately 1200 cells was targeted, with one or five ions, cellular damage measured as induced micronuclei was increased by 20%. When a fraction from 1% to 20% of cells were individually targeted, the micronuclei yield in the population greatly exceeded that predicted on the basis of the micronuclei yield when all of the cells were targeted assuming no bystander effect was occurring. However when 2-(4-carboxyphenyl)-4,4,5,5- tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO), a nitric oxide (NO)-specific scavenger was present in the culture medium, the micronuclei yields reduced to the predicted values, which indicates that NO contributes to the bystander effect. By using 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM), NO was detected in situ, and it was found that NO-induced fluorescence intensity in the irradiated population where 1% of cell nuclei were individually targeted with a single helium ion was increased by 1.13 +/- 0.02-fold (P <0.005) relative to control with approximately 40% of the cells showing increased NO levels. Moreover, the medium harvested from helium ion-targeted cells showed a cytotoxic effect by inducing micronuclei in unirradiated T98G cells, and this bystander response was also inhibited by c-PTIO treatment. The induction of micronuclei in the population could also be decreased by c-PTIO treatment when 100% of cells were individually targeted by one or two helium ions, indicating a complex interaction of direct irradiation and bystander signals.

Mature DC from skin and skin-draining LN retain the ability to acquire and efficiently present targeted antigen.

Skin-draining LN contain several phenotypically distinguishable DC populations, which may be immature or mature. Mature DC are generally considered to have lost the capacity to acquire and present newly encountered Ag. Using antibody-opsonized liposomes as Ag carriers, we show that mature DC purified from skin explants are able to efficiently capture liposomes, process Ag encapsulated within them and activate Ag-specific CD4(+) T cells. Explant DC from mice with Langerhans cells (LC) expressing the primate diphtheria toxin receptor that were exposed to diphtheria toxin in vivo presented Ag as well as explant DC from wild-type mice, indicating that LC are not required and dermal DC are probably responsible for this presentation. We further show that all DC subtypes from LN that capture opsonized Ag are capable of cross-presenting it to CD8(+) T cells. Induction of additional maturation in vivo by LPS or treatment with double-stranded RNA did not alter the Ag presentation capacity of the skin or LN DC subtypes. These results suggest that mature DC present in skin-draining LN may play an important role in the induction of primary and/or secondary immune responses against Ag delivered to the LN that they take up by receptor-mediated endocytosis.

Low-dose studies of bystander cell killing with targeted soft X rays

The Gray Cancer Institute ultrasoft X-ray microprobe was used to quantify the bystander response of individual V79 cells exposed to a focused carbon K-shell (278 eV) X-ray beam. The ultrasoft X-ray microprobe is designed to precisely assess the biological response of individual cells irradiated in vitro with a very fine beam of low-energy photons. Characteristic C-K X rays are generated by a focused beam of 10 keV electrons striking a graphite target. Circular diffraction gratings (i.e. zone plates) are then employed to focus the X-ray beam into a spot with a radius of 0.25 mum at the sample position. Using this microbeam technology, the correlation between the irradiated cells and their nonirradiated neighbors can be examined critically. The survival response of V79 cells irradiated with a C-K X-ray beam was measured in the 0-2-Gy dose range. The response when all cells were irradiated was compared to that obtained when only a single cell was exposed. The cell survival data exhibit a linear-quadratic response when all cells were targeted (with evidence for hyper-sensitivity at low doses). When only a single cell was targeted within the population, 10% cell killing was measured. In contrast to the binary bystander behavior reported by many other investigations, the effect detected was initially dependent on dose (200 mGy). In the low-dose region (