P<0.05 compared with others.
Morphological identification of apoptosis
The cells were collected and stained with Giemsa at various times after treatment with antisense oligonucleotide or/and arsenic trioxide. Apoptotic changes (blebbing, chromosome fragmentation and condensation, and formation of apoptotic bodies) were observed using an Olympus Research microscope.
DNA content analysis by flow cytometry
The cells were collected, washed twice with PBS, and fixed with 70% alcohol at 4 °C for 24 h. After two further washings, 200 μl of RNAase A (1 mg/ml) and 800 μl of propidium iodide (100 μg/ml) were added at 4 °C for 30 min. DNA content was then analyzed by flow cytometry.
Detection of bcl-2 protein by flow cytometry
Cells (5×105) were collected and fixed with 70% formaldehyde at 4 °C for 15 min, then washed and re-suspended twice in specific PBS (s-PBS) (PBS supplemented with 1% human AB serum, 1% Tween-20 from Boehringer Mannheim, pH 7.2) to permeabilize them. Bcl-2 antibody (Dako, Denmark) (50 μl) was added to the cell suspension and the mixture was incubated at 4 °C for 1 h. After two further washings with s-PBS, 50 μl FITC-IgG (2nd antibody) (Dako, Denmark) was added and incubated at 4 °C for 30 min. Finally, the cells were washed twice with PBS and the level of bcl-2 protein was determined by flow cytometry.
SPSS statistical software was used to analyze the results and P<0.05 was taken as the criterion ofsignificance.
NB4 and K562 cells treated with various concentration of arsenic trioxide showed morphological changes characteristic of apoptosis, such as cell shrinkage, chromatin condensation, fragmentation of nuclei and formation of apoptotic bodies (Fig. 1).
Effect of arsenic trioxide on leukemic cells
After NB4 and K562 cells had been treated with various concentrations of arsenic trioxide for different times, some cells had become apoptotic and growth was inhibited. Fig. 2shows that NB4 cells were more sensitive.Slight but significant inhibition of growth was observed at 0.25 μmol/l arsenic trioxide, but there was no effect on the growth of K562 cells at this concentration. At higher concentrations of arsenic trioxide (0.5 to 2 μmol/l), most of the NB4 cells were killed but only slight inhibition of K562 cells growth was observed at 2 μmol/l.
The synergistic inhibitory effects of bcl-2 antisense and arsenic trioxide on NB4 cell growth. Cells were incubated with arsenic trioxide (0.25 μmol/l), ASODN (10 μmol/l), As2O3(0.25 μmol/l)+SODN (10 μmol/l) (control), or As2O3(0.25 μmol/l)+ASODN (10 μmol/) for 24, 48 72 and 96 h. The growth of NB4 cells treated with As2O3and ASODN in combination was significantly inhibited compared with the control and other groups between 24 and 96 h (P<0.05 or 0.01).
The synergistic inhibitory effect of bcl-2 antisense and arsenic trioxide on K562 cell growth. Cells were incubated with arsenic trioxide (2.0 μmol/l), ASODN (10 μmol/l), As2O3(2.0 μmol/l)+SODN (10 μmol/l) (control), or As2O3(2.0 μmol/l)+ASODN (10 μmol/) for 24, 48 72 and 96 h. The growth of K562 cells treated with As2O3and ASODN in combination was significantly inhibited compared with the control and other groups between 24 and 96 h (P<0.05 or 0.01).
Synergetic inhibitory effects of bcl-2 antisense and arsenic trioxide on growth of leukemic cells
As shown in fig. 3, ASODN alone at 10 μmol/l had no significant effect on the growth of NB4 cells. However, there was significant inhibition from 24 to 96 h when 0.25 μmol/l arsenic trioxide and 10 μmol/l of ASODN were used in combination, and this inhibition grew more marked at longer incubation times. At 96 h, the survival rate of NB4 cells was about half of that observed when ASODN or arsenic trioxide alone were used.
As shown in fig. 4, similarly, in K562 cells, 10 μmol/l ASODN had no significant effect on growth, but there was significant inhibition from 24 to 96 h when 2.0 μmol/l arsenic trioxide and 10 μmol/l ASODN were used in combination. Again, this inhibition became more marked at longer incubation times, and at 96 h the survival rate of K562 cells was about half of that observed with ASODN or arsenic trioxide alone.
bcl-2 antisense increases the susceptibility of leukemic cells to arsenic trioxide-induced apoptosis
As shown in Table 134.67±4.4 of NB4 cells and 38.45±6.2 of K562 cells became apoptotic when arsenic trioxide and ASODN were used in combination. When arsenic trioxide or ASODN was used alone, the equivalent percentages were 5.22±1.8 and 15.68±4.2 for NB4 cells and 14.61±3.2 and 10.66±3.5 for K562 cells. The control oligonucleotide (SODN), unlike ASODN, did not affect the susceptibilities of the leukemic cells to arsenic trioxide.
Effect of bcl-2 antisense in combination with arsenic trioxide on bcl-2 protein expression
As shown in Table 2, the percentages of bcl-2 protein expressed in NB4 and K562 cells (NB4: 26.94±3.92 and K562: 20.65±5.68) were significantly when arsenictrioxide and ASODN were used in combination than when either was used singly.
The results described in this paper show that low doses of arsenic trioxide (up to 0.25 μmol/l for NB4 and 2.0 μmol/l for K562) do not affect leukemiccell growth significantly (Fig. 1). However, the susceptibility of leukemic cells to arsenic trioxide is greatly increased by bcl2-antisense oligodeoxyribonucleotides. Under the combined treatment, cell growth was inhibited, a large number of leukemic cells died, and expression of the bcl-2 gene was significantly (Figs. 3 and 4). The pharmacological effect of arsenic trioxide is therefore strengthened by bcl-2 antisense.
Although both NB4 and K562 have high levels of bcl-2 expression, they differ markedly in their susceptibility to arsenic trioxide, as shown in Fig. 2(Bai et al., 1998; Zhang and Nie, 2001). The reason for this difference in susceptibility is unclear at present.
The cytotoxic effects of arsenic trioxide are complex and involve many genes, PML/RARα and BCR/ABL genes contribute to the apoptotic processes induced by arsenic trioxide (Chen et al., 1997; Xiao et al., 1998). However, the bcl-2 gene is prominent in regulating cell growth and apoptosis, and bcl-2 antisense is known increase the susceptibility of leukemic or other cancer cells to chemotherapeutic drugs (Keith et al., 1995; Ziegler et al., 1997). The results found in this study are consistent with this view.
This research was supported by the National Natural Science Foundation of China (No. 39870361), Guangdong Science Foundation (No. 980710) and Guangdong Science Plan Foundation (No. 99M01204G).
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Received 24 January 2002/26 May 2003; accepted 8 July 2003