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Summary. Clinical efficacy of As2O3 has been shown in patients with relapsed acute promyelocytic leukaemia (APL). There is evidence that the effects of As2O3 are not restricted to events specific for APL. As2O3 might target mechanisms involved in the pathogenesis of other malignancies. We assessed susceptibility to induction of apoptosis by As2O3 and cytostatics in 22 myeloid and non-myeloid malignant cell lines. As2O3 was used in concentrations of 0·01–10 µmol/l. Cell lines displayed different kinetics of response and different sensitivity to As2O3. The minimum concentration of As2O3 for induction of apoptosis was 0·1 µmol/l. High concentrations of As2O3 (5 µmol/l) induced apoptosis in a large proportion of cells in all cell lines tested. Low (1 µmol/l As2O3) concentrations induced apoptosis in NB-4, HL-60, U-937, CEM, HL-60, KG-1a, PBL-985, ML-2 and MV-4–11, but not in HEL, K-562, KG-1 and Jurkat up to 35 d of incubation. However, the non-apoptotic population of 1 µmol/l As2O3-treated HEL, K-562, K-562 (0·02), K-562(0·1) and Jurkat showed reduced proliferation. CEM as well as its' multidrug-resistant derivatives were sensitive to 1 µmol/l As2O3. In summary, these data demonstrate that As2O3-induced apoptosis is not restricted to cell lines with t(15;17). Apoptosis was induced in vitro by As2O3 concentrations that are achievable in vivo after infusion of well-tolerated As2O3 doses. Thus, As2O3 might be a suitable therapeutic agent for malignancies other than APL provided the adequate dose and duration of As2O3 treatment are used.
Several reports have demonstrated the efficacy of As2O3 in the treatment of relapsed or refractory acute promyelocytic leukaemia (APL) with complete remission rates of > 72% (Shen et al, 1997; Soignet et al, 1998; Warrell et al, 1998; Niu et al, 1999; Spencer & Firkin, 1999; Jiong et al, 2000). As2O3 was found to be a potent alternative in treatment of all-trans retinoic acid (ATRA)-resistant APL (Chen et al, 1997; Kitamura et al, 1997). To date, the clinical use of As2O3 in leukaemia treatment has been restricted to APL because it was thought that As2O3 triggered the degradation of the t(15;17)-specific fusion protein PML-RARα (Chen et al, 1996).
However, recent reports show that As2O3 can exert PML-RARα-independent effects (Wang et al, 1998; Huang, C. et al, 1999; Huang, X.J. et al, 1999). Moreover, the potency of As2O3 to induce apoptosis was shown for non-APL leukaemia and lymphoma cell lines (Konig et al, 1997; Wang et al, 1998; Lu et al, 1999; Zhu et al, 1999; Puccetti et al, 2000; Walter et al, 2000) as well as other tumour cell lines (Akao et al, 1999; Huang, C. et al, 1999; Zhang et al, 1999; Deng et al, 2000; Chen, F. et al, 2001; Jiang et al, 2001). The necessity of the PML-RARα fusion protein present in APL-cell lines for As2O3-induced apoptosis thus became questionable. As2O3 can induce activation of caspases (Akao et al, 1999; Huang, X.J. et al, 1999; Kitamura et al, 2000; Jiang et al, 2001), downregulation of Bcl-2 (Chen et al, 1996; Deng et al, 2000; Perkins et al, 2000; Puccetti et al, 2000), modulation of p53 (Jiang et al, 2001), as well as the uncoupling of the mitochondrial potential (Dai et al, 1999; Jing et al, 1999; Kroemer & de The, 1999; Larochette et al, 1999; Cai et al, 2000). It is speculated that these effects are responsible for programmed cell death, whereas the induction of cyclin-dependent kinase inhibitors seems to play a role in As2O3-mediated cell cycle arrest and inhibition of cell proliferation (Eguchi et al, 1997; Seol et al, 1999; Park et al, 2000).
In this paper, we address the question of whether clinically achievable concentrations of As2O3 (Shen et al, 1997; Ni et al, 1998) are able to induce both apoptosis and growth inhibition in a broad spectrum of leukaemia and lymphoma cell lines without t(15;17). We therefore tested 22 cell lines that represent various stages of lympho-haemopoietic differentiation, including six cell lines resistant to different cytostatic agents. The focus is set on induction of apoptosis in long-term incubation experiments. Incubation time and As2O3 concentrations were derived from treatment duration and serum As2O3 concentrations in successful clinical trials of As2O3 in APL.
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As2O3 is being increasingly used as a successful treatment of APL (Chen, Z. et al, 2001). At first it was thought that the effects of As2O3, e.g. reduction of cell division rates and induction of apoptosis, were specific for APL cell lines. In APL, the t(15;17) translocation results in a fusion of PML and RARα. It has recently been shown that As2O3 triggers the degradation of both PML-RARα and wild-type PML (Muller et al, 1998a,b; Sternsdorf et al, 1999). The ubiquitin-like protein SUMO-1 can covalently bind to the PML and PML-RARα. This post-transcriptional modification of PML leads to a delocalization of the protein from nucleus into compartments, in which the protein is degraded. As2O3 modulates the SUMO-1 binding to PML and PML-RARα. Several reports showed evidence that the activation of caspases (Akao et al, 1999; Huang, X.J. et al, 1999; Kitamura et al, 2000; Jiang et al, 2001), modulation of p53 (Jiang et al, 2001) and uncoupling of the mitochondrial potential (Dai et al, 1999; Jing et al, 1999; Kroemer & de The, 1999; Larochette et al, 1999; Cai et al, 2000) are involved in As2O3-induced apoptosis.
These cellular effects suggest that As2O3 may induce apoptosis not only in APL cell lines. Therefore, we performed a systematic analysis of the effect of As2O3 in a great variety of non-APL cell lines representing a broad range of lympho-haemopoietic differentiation. In our report, the analysis of 22 different leukaemia and lymphoma cell lines clearly shows that induction of apoptosis as well as reduction of proliferation rate are not restricted to APL cell lines. We were able to classify 22 leukaemia and lymphoma cell lines into three groups according to their sensitivity to As2O3-induced apoptosis. All cell lines responded to As2O3 at concentrations that were shown to be achievable by administration of well-tolerated doses of As2O3 in clinical studies (Shen et al, 1997; Ni et al, 1998). As2O3 at a concentration of 0·1 µmol/l was sufficient to induce apoptosis in group A cell lines; group B cell lines became apoptotic with As2O3 concentrations ≥ 1 µmol/l. An As2O3 concentration of 5 µmol/l was necessary to induce apoptosis in group C cell lines.
The findings of apoptosis-inducing effects of As2O3 in non-APL cells are in agreement with several reports about apoptotic effects of As2O3 even in non-haemopoietic cells such as the cervical cancer cell line SiHa (Deng et al, 2000), neuroblastoma cell lines (Akao et al, 1999), head neck cancer cell line (Seol et al, 1999), gastric cell line MGC-803 (Zhang et al, 1999), hepatocarcinoma cell lines (Liu et al, 2000), prostate and ovary cancer cell lines (Uslu et al, 2000).
The incubation time necessary to induce apoptosis shows broad variation. Notably, some cell lines (e.g. HL-60, K-562, HEL, Jurkat) became apoptotic only after prolonged incubation. This should be considered when designing schedules for treatment of leukaemia or lymphomas with As2O3. In clinical trials of As2O3 in refractory APL, the median time to remission during As2O3 therapy ranged from 31 d (Niu et al, 1999) to 47 d (Soignet et al, 1998).
Patients refractory to conventional acute myeloid leukaemia (AML) treatment might be the first group considered for clinical trials of As2O3 in non-APL leukaemias. In this context, it is important to exclude the possibility that resistance to, for example, anthracyclines or topoisomerase inhibitors is also associated with resistance to As2O3. Therefore, we studied As2O3 sensitivity in cell lines resistant to camptothecin, doxorubicin and mitoxantrone.
Apoptosis could be induced in four out of six cell lines resistant to conventional cytostatic drugs in the same range as in their parental cytostatic-sensitive cell lines. Only in the case of mitoxantrone-resistant HL-60/MX1 and HL-60/MX2 cell lines was the parental cell line HL-60 more sensitive to As2O3-induced apoptosis. Thus, our in vitro results suggest that As2O3 might be effective in AML refractory to conventional cytostatic drugs.
Apart from the apoptosis-inducing effect, As2O3 seems to inhibit proliferation of all leukaemia and lymphoma cell lines tested. Cell lines such as 697, Jurkat, HL-60, U937, PBL-987, K-562, KG-1 and HEL that were less sensitive or insensitive to 1 µmol/l As2O3 showed a clear reduction of proliferation rate after 10 d at 1 µmol/l As2O3. Growth inhibition could even be observed in all derivates of K562, HL-60 which are resistant to different cytostatic drugs. As demonstrated by DNA histograms, As2O3 at concentrations of 1 µmol/l did not lead to cell cycle arrest. A potential action of As2O3 that could explain the effects on proliferation could be the inhibition of aggregation of tubulin (Li & Broome, 1999). Our cell cycle studies are in accordance with recently published data reporting that arsenic(III)-oxide in concentrations lower than 2 µmol/l does not induce G2/M growth arrest in U937 cells (Park et al, 2001).
In a murine model, the antitumour effect of As2O3 on experimental liver cancer was reported. As2O3 showed an obvious antitumour effect, caused by apoptosis induction with reduction of Bcl-2 expressing cells in the tumour mass (Chen et al, 2000). The in vivo and in vitro effects of As2O3 on a neuroblastoma mouse model were described (Ora et al, 2000). As2O3 inhibited the tumour growth and induced apoptosis.
Promising results of the clinical trials in APL, our in vitro results on As2O3-mediated apoptosis and cell proliferation, and the promising results from mouse models suggest the use of As2O3 also for refractory non-APL leukaemia. In one patient with secondary acute leukaemia we have induced partial remission by using As2O3 (unpublished observations). Thus, As2O3 might be a suitable therapeutic agent for myeloid malignancies other than APL and non-myeloid malignancies, provided an adequate dose and duration of As2O3 treatment are used. We plan a pilot study for patients with refractory AML who are not eligible for allogeneic stem cell transplantation.