The staining procedure using the monoclonal antibody to BrdUrd involved the following important steps: the duration of BrdUrd labeling time, the concentration of HC1, the duration of HCI treatment for cellular DNA partial denaturation and so on. The ratio of mean intensities between BrdUrd positive cells and BrdUrd negative cells, the proportion of cell aggregation occuring during HCI treatment, the relative fluorescence intensity and coefficient of variation (CV) of G_1 peak were considered as the criteria of optimal conditions of the whole staining procedure. The optimal results of this staining procedure were obtained under the conditions of 30 rain BrdUrd labeling time, 2.4 mol/L HC1, 30 min HC1 treatment, 1 hour incubation of the monoclonal antibody to BrdUrd and without RNase treatment. With this staining procedure, the optimal staining results were obtained for KF-1, KFr, HeLa and IK-90 cell lines.

OBJECTIVETo explore the association of inherent cellular reactive oxygen species (ROS) levels with susceptibility of the tumor cells to apoptosis induction by arsenic trioxide (As(2)O(3)).

METHODSLow concentration (2 micromol/L) of As(2)O(3) was administered to two cultured leukemic cell lines, NB4 and U937, and two esophageal carcinoma cell lines, EC1.71 (also named EC/CUHK1) and EC1867, to confirm the difference in apoptosis susceptibility of NB4 versus U937 and of EC1.71 versus EC1867. Dihydrogenrhodamine 123 (DHR123), used as a ROS capture agent, was incubated with cells in the absence of As(2)O(3). Fluorescence intensity of rhodamine 123, the product of cellular oxidation of DHR123, was detected by flow cytometry and ROS was measured.

RESULTSLow concentration of As(2)O(3) induced apoptosis was more likely to occur in NB4 and EC1.71 cells than in U937 and EC1867 cells, or NB4 was more sensitive than U937, and EC1.71 more sensitive than EC1867 to As(2)O(3). The inherent cellular ROS level is higher in NB4 than in U937, and also higher in EC1.71 than in EC1867.

CONCLUSIONSThe difference in cellular ROS level is positively associated with cellular susceptibility to apoptosis induction by As(2)O(3). The inherent ROS level might be important in defining apoptotic susceptibility to As(2)O(3).

Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; genetics ; Arsenicals ; pharmacology ; DNA, Neoplasm ; genetics ; Flow Cytometry ; Fluorescent Dyes ; Humans ; Oxides ; pharmacology ; Reactive Oxygen Species ; metabolism ; Rhodamine 123 ; Tumor Cells, Cultured ; drug effects ; metabolism

OBJECTIVETo investigate whether ascorbic acid could enhance the efficacy of arsenic trioxide (As(2)O(3)) combined with 2, 3-dimethoxy-1, 4-naphthoquinone (DMNQ) in inducing the apoptosis of leukemia cell line U937 and its possible mechanism.

METHODSFlow cytometry and electron microscopy were applied to detect apoptosis of U937 cells after treatment with various combinations of As(2)O(3), DMNQ and ascorbic acid for 24 hours.

RESULTSAs(2)O(3) and DMNQ induced-apoptosis of U937 cells was enhanced (35.24%-->61.20%) upon cotreatment with ascorbic acid. Catalase could reverse this effect of DMNQ. Ascorbic acid had no effect on DMNQ-induced apoptosis of U937 cells.

CONCLUSIONAscorbic acid enhanced the apoptosis of U937 cells via reactive oxygen species-dependent pathway in the presence of As(2)O(3).

Apoptosis ; drug effects ; Arsenicals ; pharmacology ; Ascorbic Acid ; pharmacology ; Drug Synergism ; Flow Cytometry ; Humans ; Naphthoquinones ; pharmacology ; Oxides ; pharmacology ; U937 Cells