Measurement of potential doubling time for human tumor xenografts using the cytokinesis-block method.

Estimates of the potential doubling time (Tpot) of seven human tumor xenografts were made using a cytokinesis-block method. This method is currently being investigated as an alternative to flow cytometric assays using the administration of a thymidine analog in the measurement of Tpot. If perfected, the cytokinesis-block method of measuring Tpot would be advantageous as a predictive assay, in that no label is administered to the tumors in situ. Xenografts were grown in nude mice, and following tumor excision and disaggregation, tumor cells were cultured with the cytokinesis-blocking agent cytochalasin B. The flux of cells through mitosis was marked by the accumulation of multinucleate cells. By counting the total number of nuclei as a function of time, the effective population growth was observed. Tpot values were obtained by fitting suitable exponential least squares curves to the data, with the doubling time indicated by the fitted functions. For the seven tumors studied, a significant spread in growth rates was observed. Tpot values generated by this method ranged from approximately 2 days for a rapidly growing squamous cell carcinoma of the pharynx, FaDu, to approximately 7.5 days for the slower growing glioblastoma multiforme U251-MG. These values are compared with standard 5-iododeoxyuridine Tpot measures and volume doubling times obtained by Perez et al. (Cancer Res., 55: 392-398, 1995) for the same tumor xenografts. Although individual Tpot values varied between these methods, the ranking of the seven tumors in order of Tpot times was the same regardless of method. In addition to estimating Tpot, for each of the tumors, the fraction of clonogenically dead cells that was microscopically apparent, including apoptotic cells and cells expressing micronuclei, was determined as a function of time in culture. Tracking this in vitro cell loss rate provides information on the adjustment of these primary tumor cells to in vitro culture, a factor that needs to be addressed when determining how in vitro measurements of Tpot can be effectively related to in vivo measurements.

Long-term ascorbic acid administration reverses endothelial vasomotor dysfunction in patients with coronary artery disease.

BACKGROUND: Loss of endothelium-derived nitric oxide (EDNO) contributes to the clinical expression of coronary artery disease (CAD). Increased oxidative stress has been linked to impaired endothelial vasomotor function in atherosclerosis, and recent studies demonstrated that short-term ascorbic acid treatment improves endothelial function. METHODS AND RESULTS: In a randomized, double-blind, placebo-controlled study, we examined the effects of single-dose (2 g PO) and long-term (500 mg/d) ascorbic acid treatment on EDNO-dependent flow-mediated dilation of the brachial artery in patients with angiographically established CAD. Flow-mediated dilation was examined by high-resolution vascular ultrasound at baseline, 2 hours after the single dose, and 30 days after long-term treatment in 46 patients with CAD. Flow-mediated dilation improved from 6.6+/-3.5% to 10.1+/-5.2% after single-dose treatment, and the effect was sustained after long-term treatment (9. 0+/-3.7%), whereas flow-mediated dilation was 8.6+/-4.7% at baseline and remained unchanged after single-dose (7.8+/-4.4%) and long-term (7.9+/-4.5%) treatment with placebo (P=0.005 by repeated-measures ANOVA). Plasma ascorbic acid concentrations increased from 41.4+/-12. 9 to 115.9+/-34.2 micromol/L after single-dose treatment and to 95. 0+/-36.1 micromol/L after long-term treatment (P<0.001). CONCLUSIONS: In patients with CAD, long-term ascorbic acid treatment has a sustained beneficial effect on EDNO action. Because endothelial dysfunction may contribute to the pathogenesis of cardiovascular events, this study indicates that ascorbic acid treatment may benefit patients with CAD.

The synthesis of nucleoside 5'-O-(1,1-dithiotriphosphates).

Appropriately protected nucleoside 5'-O-(2-thio-1,3,2-dithiaphospholanes) react with inorganic pyrophosphate in the presence of a strong base catalyst (DBU) to give nucleoside 5'-O-(1,1-dithiotriphosphates) 1a-g. The latter compounds, including an AZT analogue, show modest antivirial activity against HIV-1 and HIV-2 replication in CEM cells. The AZT and deoxyadenosine derivatives were found to be inhibitors of HIV reverse transcriptase.

Synthesis and interaction with thymidylate synthase of 5'-dithiophosphate and 5'-fluorothiophosphate of thymidine.

Thymidine-5'-fluorothiophosphate, dTMP(S)-F, was synthesized by the oxathiaphospholane, and thymidine 5'-dithiophosphate, dTMPS2, by the dithiaphospholane, method. To estimate the role of 5'-phosphate group ionization in binding of pyrimidine nucleotides by thymidylate synthase, dTMP(S)-F was studied as an inhibitor of mouse tumour (L1210) enzyme, and its inhibitory properties were compared with those of dTMPS2, a close dTMP analogue. While dTMPS2 proved to be an inhibitor, competitive vs dUMP, with K(i)app = 94 microM, the 5'-fluorothiophosphate congener displayed no activity, indicating that the enzyme requires for binding the presence of a dianionic 5'-phosphate group in a nucleotide.