he HPLC columns were 18 kept at 27.8C. The mobile phase was delivered at a flow-rate of 1.0 ml/min, with the following stepwise gradient elution program: A-B (60:40) at 0 min→(40:60) at 30 min→(40:60) at 60 min. Solvent A contained 10 mM tetrabutylammonium hydroxide, 10 mM KH PO and 0.25% MeOH, and was adjusted to pH 6.9 with 1 M HCl. Solvent B consisted of 5.6 mM 24 tetrabutylammonium hydroxide, 50 mM KH PO and 30% MeOH, and was neutralized to pH 7.0 with 1 M NaOH. The 24 calibration curves (r.0.99) of the components in cell extracts were established with their aqueous standards. The average within-day precision for the nine compounds was 0.9%, and the average day-to-day precision was 5.0%. The detection limits (pmol) of the nine reagents were 1.39 (ADP), 4.32 (CTP), 15.5 (dCTP), 2.38 (GTP), 4.42 (UTP), 9.45 (dGTP), 14.6 (dTTP), 2.44 (ATP) and 11.8 (dATP). The recovery of this method for the standards ranged from 82.4 to 120.5%. The results for the detection of nucleotide pools in 16 normal and tumor cell lines were presented. In conclusion, this simplified analytical method enables the simultaneous quantitation of NTP and dNTP in cell or tissue extracts and may represent a valuable tool for the detection of minute alterations of intracellular NTP/dNTP pools induced by anticancer/antiviral drugs and diseases.

We constructed a biologicmaterial composedoffetal ratbrain cells (neurospheres) and adult rat bone marrow stromal cells (MSCs), designated as NMCspheres Adult rats were subjected to 2 hours of middle cerebral artery occlusion (MCAo) and implanted with cultured prelabeled NMCspheres (n=6), neurospheres (n=5) or MSCs (n=6) into the ischemic penumbra at 24 hours after MCAo. Control adult rats (n=10) were subjected to MCAo alone. In vitro within the NMCspheres, MSCs rapidly fonned a process network with intact neural cells compared with a necrotic core within neurospheres alone An in vivo rat corneal assay demonstrated that NMCspheres enhanced angiogenesis compared to MSCs and neurospheres Neurological functional recovery after stroke was enhanced in rats trcated with NMCspheres, compared to rats with neurosphere or MSC treatments by day 7 and day 14 after transplantation. The NMCspheres are a new compositc material that may be employed in the treatment of stroke

(E)-2'-Deoxy-(fluoromethylene) cytidine (FMdC) is known as an inhibitor of ribonucleoside diphosphate reductase, a key enzyme in the de novo pathway of DNA synthesis. FMdC was tested as a modifier of radiation response in vitro on a human colon carcinoma cell line (WiDr), and the observed radiosensitization was confirmed on two human cervix cancer cell lines (C33-A and SiHa). Using the clonogenic assay, the effect ratio (ER) at a clinically relevant dose level of 2 Gy was 2.10 (50nM FMdC), 1.70 (30nM FMdC), and 1.71 (40nM FMdC) for the three cell lines WiDr, C33-A, and SiHa, respectively. A more detailed analysis of the importance of timing and concentration of FMdC was done on the WiDr cell line alone, yielding an increased ER (2Gy) with increasing concentration and duration of exposure to the drug, ranging from 1.0 (6h) to 1.8 (72h) at 30 nM FMdC and from 1.2 (6h) to 3.5 (24h) at 300nM. We investigated the effect of FMdC on the cellular deoxynucleotide triphosphate pool in WiDr cells and demonstrated a marked depletion of dATP and a significant rise of TTP levels. Cell cycle analysis showed early S-phase accumulation induced by FMdC alone, G2-M block induced by irradiation alone, and an increased accumulation of cells in G2-M if both modalities are used. Our data suggest that FMdC is a radiation response modifier in vitro on different cancer cell lines. The observed radiosensitization may in part be explained by alteration of the deoxynucleotide triphosphate pool, which is consistent with the effect of FMdC on ribonucleoside diphosphate reductase.

Three novel taxinine analogues were prepared and tested for their activity as multidrug resistance (MDR) reversal agents in comparison with verapamil. In vitro testing demonstrated that compounds 8-10 possess MDR-reversal activity in the KB/V cell line. Half-hour after treatment with 5, 10, and 20lmol/L compound 9, the intracellular rhodamine123 concentration increased 2.3, 2.9, and 3.2-fold, respectively, higher than 1.88-fold of 10lmol/L verapamil in KB/V cell line. In vivo studies with VCR-resistant KB/V tumor xenografts showed that compound 9 in combination with VCR significantly inhibited tumor growth. Treatment with VCR or 9 alone did not result in growth inhibition. These results reveal that three taxinine analogues are good modifiers of MDR in tumor cells.