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Keywords:

  • leukaemia;
  • arsenic trioxide;
  • apoptosis;
  • caspases;
  • Bcl-2

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

We showed that arsenic inhibited the cell growth of four B-cell leukaemia cell lines of 11 various cell lines in vitro. In two of these four lines, KOCL44 and LyH7, apoptosis was identified by morphological and nucleosomal DNA fragmentation studies. Three of the four B-cell lines that were growth inhibited were acute infantile leukaemia with t(11;19)(q23;p13) translocations involving the MLL gene that encodes the transcriptional factor Drosophila trithorax. The arsenic-induced apoptosis in KOCL44 and LyH7 cells was found to be linked to caspases by Western blot and enzymological analyses. The amount of Bcl-2 was reduced during apoptosis in LyH7 as judged by Western blot analysis. We concluded that combined activation of the caspases and down-regulation of Bcl-2 could determine the fate of B-cell leukaemic cells in response to arsenic.

Recently, it was reported by Chen et al (1996) that arsenic trioxide (As2 O3) is very cytotoxic to acute promyelocytic leukaemia (APL) cells, which carry the specific t(15;17)(q22;q11) chromosomal translocations in > 70% of patients, by in vitro study. This study originated from clinical experience indicating that As2O3 is very effective in APL patients, especially in cases which non-responsive to all-trans-retinoic acid (ATRA) or other chemotherapeutic drugs ( Shen et al, 1997 ). As for the mechanism of the effect of As2O3 on APL cells, arsenic has been reported to cause apoptosis directly through the down-regulation of Bcl-2 in NB4 cells of APL ( Chen et al, 1996 ). It was also suggested from an immunocytochemical study that As2O3 functions as a modulator of chimaeric PML/RARα protein, which could be directly responsible for its leukaemogenic potential ( Chen et al, 1996 ).

Arsenic compounds have been accepted as responsible for lung cancer, skin cancer and other diseases. The use of arsenic compounds as drugs has a long history in Chinese traditional medicine, and fortunately arsenic does not produce any myelosuppression in most patients receiving As2O3 treatment ( Shen et al, 1997 ).

Considering the mechanism that arsenic induces apoptosis by the down-regulation of Bcl-2, arsenic may inhibit cell growth not only of APL cells but also of other kinds of leukaemias. In this study we exposed various kinds of leukaemic cells to As2O3 and elucidated the mechanism of its action. We found that arsenic-induced apoptosis in B-cell leukaemia cell lines, KOCL44 and LyH7, occurred through the involvement of interleukin-1β converting enzyme (ICE) family proteases (the caspases), such as caspase 1 (ICE) and caspase 3 (CPP32/Yama). In LyH7 cells the down-regulation of Bcl-2 was found.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

Chemicals

An As2O3 preparation (Sigma, St Louis, Mo.) was used. Stock solution was made at the concentration of 1 m M with PBS and diluted to the working concentration before use.

Cell culture, cell viability, and morphological study

Human and mouse leukaemia cell lines were grown in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum (CSL Ltd, Victoria, Australia) and 2 m ML-glutamine in an atmosphere of 95% air and 5% CO2 at 37°C.

The cell lines used in this study were: HL60 (human promyelocytic leukaemia); K562 (human monocytic leukaemia); RC-K8 (human B-cell lymphoma with t(11;14) (q23;q32)) ( Akao et al, 1990); KOPN-1 , KOCL33 and KOCL44 [human infantile B-cell leukaemia with t(11;19)(q23;p13)] ( Iida et al, 1992 ); ATN-1 (human adult T-cell leukaemia) ( Naoe et al, 1988 ); Meg-01 (human megakaryocytic leukaemia); NP3 (mouse B-cell plasmacytoma); LyH7 (mouse B-cell leukaemia) ( Palacios et al, 1987 ) kindly donated by Dr Ronald Palacios, University of Texas; and WEHI-3B (mouse B-cell leukaemia).

Cell viability was determined by the trypan blue dye-exclusion assay. Cells were suspended at the density of 5 × 105/ml in fresh medium and treated with As2O3.

For morphological examination, cells were stained with Hoechst 33342 (5 μg/ml) at 37°C for 30 min, washed twice with PBS, pipetted dropwise onto a glass slide, and examined by fluorescence microscopy using an Olympus microscope (Tokyo) equipped with an epi-illuminator and appropriate filters.

Analysis of DNA fragmentation by agarose gel electrophoresis

Cellular DNA was extracted from whole cells as previously described ( Akao et al, 1990 ). RNase was added to the DNA solution at the final concentration of 20 μg/ml, and the mixture was incubated at 37°C for 30 min. Electrophoresis was performed on a 2.5% agarose gel. After electrophoresis, DNA was visualized by ethidium bromide staining.

Western blot analysis

Before and after treatment with As2O3, cells were washed twice with PBS, lysed in lysis buffer (2 × PBS, 0.1% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1 m M phenylmethanesulphonyl fluoride), and then homogenized with an ultrasonic homogenizer (Heat Systems-Ultrasonics, Farmingdale, N.Y.). To condense early apoptic cells from 24 h and 48 h treated LyH7 cells, we performed the density centrifugation using Ficoll-Paque (Pharmacia, Tokyo) for blot of Bcl-2 expression. Homogenized samples were used without centrifugation. The protein concentration was determined by the method of Markwell et al (1981 ). 20 μg of protein from the homogenized sample or a sample with the same number of cells was separated by SDS-PAGE using a 12% polyacrylamide gel and electroblotted onto a PVDF membrane (Du Pont, Boston, Mass.). After blockage of nonspecific binding sites for 1 h by 5% nonfat milk in TPBS (PBS and 0.1% Tween 20), the membrane was incubated overnight at 4°C with anti-human Bcl-2 (100) antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.), anti-mouse Bcl-2 antibody (Biomol Research Laboratories, Plymouth Meeting, Pa.), anti-human caspase 1 monoclonal antibody (Upstate Laboratories, Lake Placid, N.Y.), anti-mouse caspase 1 monoclonal antibody (Santa Cruz Biotechnology), anti-human caspase 3 monoclonal antibody (Transduction Laboratories, Lexington, Ky.), or recombinant anti-phosphotyrosine antibody (Transduction Laboratories). The membrane was then washed three times with TPBS, incubated further with alkaline phosphatase-conjugated goat anti-mouse antibody (Promega, Madison, Wis.) or rabbit antibody (New England Biolabs, Beverly, Mass.) at room temperature, and then washed three times with TPBS. The immunoblot was visualized by use of an enhanced chemiluminescence detection kit (New England Biolabs).

Activities of caspase 1-like and caspase 3-like proteases and inhibition of apoptosis by their inhibitors

The activities of caspases were measured as described ( Shimizu et al, 1996 ). Briefly, cells were treated with As2O3. At the indicated times, harvested cells were suspended in 50 m M tris(hydroxymethyl) aminomethane-HCl (pH 7.4), containing 1 m M EDTA and 10 m M ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, and incubated at 37°C for 10 min in the presence of 10 m M digitonin. The lysate containing 40 μg protein was incubated with 10 μM of substrate acetyl- L-tyrosyl- L-valyl- L-alanyl- L-aspartic acid 4-methyl-coumaryl-7-amide (Ac-YVAD-MCA) (Peptide Institute, Osaka) or acetyl- L-aspartyl- L-glutamyl- L-valyl- L-aspartic acid 4-methyl-coumaryl-7-amide (Ac-DEVD-MCA) (Peptide Institute) at 37°C for 1 h. Levels of released 7-amino-methyl coumarin (AMC) were measured with a Hitachi F-3000 spectrofluorometer with excitation at 380 nm and emission at 460 nm. One unit was defined as the amount of enzyme required to release 1 pmol AMC per min at 37°C. For the study of inhibition, the tetrapeptide caspase 1-like protease inhibitor acetyl- L-tyrosyl- L-valyl- L-alanyl- L-aspartic aldehyde (Ac-YVAD-CHO) (Peptide Institute) and the tetrapeptide caspase 3-like protease inhibitor acetyl- L-aspartyl- L-glutamyl- L-valyl- L-aspartic aldehyde (Ac-DEVD-CHO) (Peptide Institute) were added 12 h before As2O3 treatment. Optimal concentrations of these inhibitors were determined from dose–response curves for the extent of cell death.

Rescue of arsenic-induced apoptosis by Ac-YVAD-CHO and Ac-DEVD-CHO was also evaluated by comparing the percentage of the number of survived cells to that of apoptic cells without inhibitors.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

We studied the growth and survival of various kinds of non-APL 11 leukaemic cell lines by treatment with As2O3 at the concentration of 0.1 or 1 μM. The concentrations of As2O3 used in this study were selected based on data reported ( Chen et al, 1996 ). At the concentration of 0.1 μM As2O3, no growth inhibition was observed in any of the cell lines, in comparison with the control before 4 d. However, the growth rate of four B-cell leukaemic cell lines, KOPN-1, KOCL33, KOCL44 and LyH7, was significantly inhibited at the concentration of 1 μM within 2 d after the start of treatment (Fig 1). It is to be noted that three, i.e. KOPN-1, KOCL33 and KOCL44, out of these four cell lines have t(11;19)(q23;p13) chromosomal translocations involving the MLL gene that encodes the transcriptional factor Drosophila trithorax ( Yamamoto et al, 1993 ).

image

Figure 1.  μM As2O3 to that in non-treated cultures. Each value represents the mean of the results obtained in two independent experiments. The leukaemic cell lines tested are indicated.

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Although arsenic inhibited the growth of four B-cell leukaemia cell lines, the nucleosomal fragmentation was demonstrated in DNAs from only KOCL44 and LyH7 cells treated with As2O3 for 2 d and 1 d, respectively ( Figs 2A and 2B). DNA fragmentation was observed to be dose dependent (data not shown). The morphological study by Hoechst 33342 staining showed the characteristic findings of apoptosis such as chromatin condensation and nuclear segmentation in all four cell lines, i.e. KOPN-1, KOCL33, KOCL44 and LyH7. The Hoechst 33342 staining images on KOCL44 and LyH7 treated with arsenic are shown in Figs 3A and 3B, respectively. Thus, arsenic-induced apoptosis was confirmed for these four cell lines. Even LyH7 cells, which overexpress Bcl-2 protein, were induced to apoptosis by exposure to As2O3 at the concentration of 1 μM for 1 d ( Figs 2B and 3B).

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Figure 2. O3 for 48 h.

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Figure 3. for 48 h and for 24 h, respectively.

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To understand the mechanism of arsenic-induced apoptosis, the time course of the expression of caspases during apoptosis in LyH7 and KOCL44 cell lines was studied by Western blot analysis. In LyH7 cells the 20 kD active form of mouse caspase 1 appeared when apoptosis was induced in the cells by treatment with As2O3 for 2 d (Fig 4A). Also, the 32 kD proenzyme and 20 kD active form of caspase 3 was shown by Western blot analysis upon arsenic-induced apoptosis in KOCL44 (Fig 4B). The amount of proenzyme decreased with the appearance of the 20 kD active form. However, the active form of caspase 1 was not detected in KOCL44 (data not shown).

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Figure 4. 5 kD) and p32 (32 kD) proenzymes, respectively, and their p20 (20 kD) active forms.

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The activities of caspase 1-like and caspase 3-like proteases during arsenic-induced apoptosis in KOCL44 cells and LyH7 cells were examined with fluorogenic tetrapeptide substrates for the same samples used for Western blot analysis. Transient increase in both protease activities was observed in KOCL44 and LyH7 cells. The activities of caspase 3-like proteases were significantly higher than those of caspase 1-like proteases by 102–103 fold. The presence of tetrapeptide protease inhibitors effectively blocked the activation of the respective proteases during arsenic treatment (Fig 6). Furthermore, in LyH7 cells the apoptic cell death was inhibited by both protease inhibitors in a dose-dependent manner up to 100 μM (Fig 5). These findings indicated that the caspases were involved in the cell death of arsenic-induced apoptosis in B-cell lines KOCL44 and LyH7.

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Figure 5. Fig 6. Activation of caspase 1-like and caspase 3-like proteases during arsenic-induced apoptosis. (A) and (C) Activity of caspase 1-like proteases. (B) and (D) Activity of caspase 3-like proteases. KOCL44 (A and B) and LyH7 (C and D) cells were treated with 1 μM As2O3. Activities of caspase 1-like and caspase 3-like proteases were measured at indicated times. Solid circles: treatment with As2O3; open circles: treatment with protease inhibitors (each 50 μM) 12 h before the addition of As2O3. Results are represented as means ± SD of values obtained from four independent experiments.

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image

Figure 6. Fig 5. Rescue of arsenic-induced apoptosis by inhibitors of caspases in LyH7 cells. The inhibitors, Ac-YVAD-CHO for caspase 1-like proteases and Ac-DEVD-CHO for caspase 3-like proteases, were both added 12 h before As2O3 exposure. The rescue of cell death was evaluated at 2 d after As2O3 exposure and shown by the percentage of the number of survived cells at each concentration of inhibitors to that of dead cells without inhibitors. Results are expressed as mean values obtained from three independent experiments.

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We next investigated the time course of Bcl-2 expression during the apoptosis in LyH7 cells, which overexpress Bcl-2 and are very sensitive to As2O3, by Western blot analysis. When the homogenates containing the same amount of protein were charged, a slight reduction in the amount of Bcl-2 during apoptosis was seen in 48 h treated LyH7 (Fig 7A). However, it was found that the amount of Bcl-2 protein was decreasing in the late apoptic LyH7 cells. When the homogenates corresponding to the same number of trypan blue-non stained early apoptic cells, which were collected by the density centrifugation, were applied, the amount of Bcl-2 per cell was found to be decreased clearly in 24 h and 48 h treated LyH7 cells (Fig 7B). This indicated that the amount of Bcl-2 protein had already decreased in the early apoptic LyH7 cells.

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Figure 7. cells collected by density centrifugation using Ficoll-Paque was > 90% and the amount of protein charged was approximately the same in three lanes. Lanes 1–3 as in (A).

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Thus, the apoptosis induced by As2O3 in LyH7 cells was linked to the activation of caspases and the down-regulation of Bcl-2.

It is well known that arsenic interferes with the phosphorylation of tyrosine. Arsenic might thus block phosphorylation of proteins associated with signal transduction. Therefore the time course of the amounts of proteins with phosphorylated tyrosines was examined for KOCL44 and LyH7 cells with anti-phosphotyrosine antibody by Western blot analysis. We found that none of them showed any significant change in amount (data not shown). Selenium, which seems to be an antagonist of arsenic, did not interfere with the cell growth inhibitory effect of arsenic in our study (data not shown).

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

The present study has shown that treatment of four B-cell leukaemic cells with As2O3 at the concentration of 1 μM caused growth inhibition and induced apoptosis. In two of them apoptosis was found to involve caspases and one of these two, LyH7, also involved Bcl-2. Although ATRA causes APL cells to differentiate and stop uncontrollable dividing, arsenic appears to work by a much different mechanism to that of ATRA. It seems to induce apoptosis without differentiation at the concentration of 1 μM in NB4 cells of APL, by the mechanism of not only modulating the chimaeric PML/RARα protein but also hindering the activity of Bcl-2, which blocks apoptosis when activated in tumour cells ( Chen et al, 1996 ).

Interestingly, three out of four B-cell lines that were susceptible to growth inhibition by arsenic carried 11q23 translocations involving MLL ( Yamamoto et al, 1993 ). It has been shown that arsenic can inactivate some important enzymes by binding to sulphydryl groups, and further induce chromosome abnormalities, sister chromatid exchanges, DNA-protein cross-links, and protein-associated DNA strand breaks in mammalian cells. Arsenic might work on the binding to DNA of transcription factors like MLL protein. Recently, it has been reported that arsenic targets PML protein, which is also a transcription factor, and induces the degradation of PML/RARα protein by the mechanism different from that of ATRA ( Zhu et al, 1997 ). This fact supports the possibility that arsenic acts on the MLL-translocation-related chimaeric protein.

We demonstrated by Western blot, enzymological, and cell death inhibition analyses that the activation of caspases was involved in arsenic-induced apoptosis, and suggested that arsenic could work upstream of the caspases to enhance the activity of caspases in both KOCL44 and LyH7 B-cell lines.

The apoptosis induced by arsenic in a B-cell line LyH7 overexpressing Bcl-2 was shown by Western blot analysis to be associated with the down-regulation of Bcl-2. In the follicular non-Hodgkin lymphomas carrying t(14;18) translocation, the overexpression of Bcl-2 protein is responsible for tumourigenesis. Recently, it has been reported that the down-regulation of Bcl-2 by antisense oligonucleotide against bcl-2 caused cell growth inhibition and improved the clinical parameters in these cases ( Webb et al, 1997 ). This finding raises the possibility that arsenic might be a possible candidate for therapy against chemotherapy-resistant B-cell malignancies.

Therefore it seems that the combination of down-regulation of Bcl-2 and activation of caspases might be essential to arsenic-induced apoptosis of B-cell leukaemic cells. This finding is similar to the fact that both Ced-3 and mammalian ICE induce apoptosis when overexpressed in a variety of nematode cells including Caenorhabditis elegans, and their proapoptotic effect can be prevented by the nematode ced-9, whose mammalian counterpart is bcl-2 ( Miura et al, 1993 ).

It seems worthwhile to expose other kinds of tumour cells to arsenic. Further studies which unveil the mechanisms of arsenic action in the induction of apoptosis will enable better clinical utilization of arsenic.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

This work was supported by a Grant from the Children's Cancer Association of Japan and a Grant-in-Aid from the Programme for Scientific Research of Ministry of Education, Science, Sports, and Culture of Japan.

References

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References
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