These results show the value of two different BAT as cellular tests in the in vitro diagnosis of patients with antibiotic allergy with equal specificity and a slightly higher sensitivity for the Flow2 CAST.

In this study we evaluate the antitumour activity, the cell cycle arrest and apoptotic properties of novel lipophilic benzene-fused seven-membered 5-fluorouracil (5-FU) analogs in comparison to 5-FU on MCF-7 human breast cancer cells. The lipophilicities of ESB-786B, ESB-252A and ESB-928A were predicted by using the CDR option of the PALLAS 2.0 program. Cytotoxic assays were evaluated in MCF-7 cells treated with the sulforhodamine B colorimetric method. Cell cycle perturbations were studied by flow cytometry. Apoptosis was determined by both DNA fragmentation and annexin V-FITC and propidium iodide staining. The novel derivatives were more lipophilic than 5-FU and induced a marked growth inhibition, in a dose-dependent manner. After treatment with IC(50) value (ranged from 2.5 to 22 microM) for each compound, light microscopy observation showed modifications in the morphology of MCF-7 cells. In addition, the 5-FU analogs arrest cells in the G(0)/G(1) phase of the cell cycle whereas 5-FU induced arrest in S-phase. Moreover, induction of apoptosis was demonstrated by the annexin-V-based assay and confirmed using DNA fragmentation analysis on MCF-7 cells, a cell line in which the induction of DNA laddering is very difficult. The novel benzannelated seven-membered 5-FU analogs can be considered as specific apoptotic inducers. These experimental findings provide support for the use of these novel compounds as new weapons in the fight against breast cancer.

No abstract

The anticarcinogenic potential of (RS)-1-(2,3-dihydro-5H-1,4-benzodioxepin-3-yl)uracil (DBDU), with the naturally occurring pyrimidine base uracil, is reported against the MCF-7 cancer cell line. The arrest in the G0/G1 and G2/M cell cycle phases was accounted for by decrease in the expression of the cyclin D1 and Cdk1 proteins, and increase in p21 and p27 proteins. Using a reverse transcription-polymerase chain reaction-based assay at a dose of 5 muM of DBDU cyclin D1 mRNA was decreased, suggesting that DBDU exerts its regulatory action on cyclin D1 at the level of transcription. DNA fragmentation was performed and demonstrated that apoptosis occurred in the tumor cell line treated with DBDU. The G0/G1 arrest is an irreversible process and the cells undergo apoptosis in a p53-independent manner. DBDU administered intravenously twice a week (50 mg/kg dose each time) induced neither toxicity nor death in mice for 5 weeks.

Attention is increasingly being focussed on the cell cycle and apoptosis as potential targets for therapeutic intervention in cancer. Taking 1-[(2-oxepanyl)]-5-fluorouracil previously prepared by us, we committed ourselves to increase the lipophilicity of this upper cyclohomologue of Ftorafur and prepared a series of bioisosteric benzannelated seven-membered 5-FU O,N-acetals to test them against the MCF-7 human breast cancer cell line. Benzo-fused seven-membered O,O-acetals or their acyclic analogues led to the expected 5-FU O,N-acetals (or aminals), in addition to six- and to 14-membered aminal structures and acyclic compounds. All the cyclic aminals provoked a G(o)/G(1)-phase cell cycle arrest, whereas Ftorafur, a known prodrug of 5-FU, and 1-[2-(2-hydroxymethyl-4-nitrophenoxy)-1-methoxyethyl]-5-fluorouracil (51) induced an S-phase cell cycle arrest. Although breast cancer is most often treated with conventional cytotoxic agents it has proved difficult to induce apoptosis in breast cancer cells and, consequently, improved clinical responses may be obtained by identifying therapies that are particularly effective in activating apoptosis. 1-(2,3-Dihydrobenzoxepin-2-yl)-5-fluorouracil (26) may be particularly useful in stimulating apoptosis in breast cancer. This compound is more potent as an apoptotic inductor than paclitaxel (Taxol). Finally, a fact that is worth emphasizing is that the cyclic and acyclic 5-FU O,N-acetals induce neither toxicity nor death in mice after one month's treatment when administered intravenously twice a week, with a 50 mg/kg dose each time. Taken together, the experimental findings provide evidence of specific anti-tumour activity of these new substances and warrant further evaluation in in vivo models of breast cancer to future clinical applications.

We appreciate the comments by Dr Monneret about the staining and gating strategies in optimizing the identification of basophils to our study on the newly developed basophil activation protocol using CD63 and CCR3 [1]. He points out that CCR3 as an identification marker for basophils is also expressed in T cells (Th2 lymphocytes and regulatory T cells), thus reducing the purity of basophils and lowering their sensitivity. According to the information of the manufacturer [2] a contamination of CD3 cells in the CCR3 population occurs with a mean of 3.85% (95% CI = 2.52-5.18%; n = 8). Furthermore, in CD4 1 CD25 1 T cells, CCR3 expression appears not to exceed 5% ( fig. 1 of [3]). If we would have these 5% T cells in the CCR3 population, they would not express CD63. Therefore an activation of, e.g., 90% CD63 would be in fact only 85.5% and an activation of 15% would be 14.25% activation. These differences might be negligible for clinical purposes. Therefore we are not in accordance with the argument that staining only with CCR3 is an insufficient step for the identification of basophils. Nevertheless, we agree with Monneret, that an additional antibody labelling T cells based on CD3 expression would be an optimal staining strategy. As the Flow2 CAST was developed for clinical routine testing, the existing protocol with two antibodies is easier to handle, particularly if studies of different labs are compared. Staining with a third antibody might be the source of additional errors and cannot be performed with older flow cytometers still in use. References1 Eberlein B, Suárez IL, Darsow U, Ruëff H, Behrendt H, Ring J. A new basophil activation test using CD63 and CCR3 in allergy to antibiotics. Clin Exp Allergy 2010; 40:411-8. 2 Test Brochure Flow2 CAST s , BÜHLMANN Laboratories AG, Schönenbuch, Switzerland. Available at http://www.buhlmann labs.ch/core/allergy/flow2-cast/ 3 Ahern D, Lloyd CM, Robinson DS. Chemokine responsiveness of CD41CD251 regulatory and CD41CD25À T cells from atopic and nontopic donors. Allergy 2009; 64:1121-9. c 2010 Blackwell Publishing Ltd, Clinical & Experimental Allergy, 40 : 953-954 954 B. Eberlein et al