Apoptotic death induced by the cyclophosphamide analogue mafosfamide in human lymphoblastoid cells: Contribution of DNA replication, transcription inhibition and Chk/p53 signaling

https://doi.org/10.1016/j.taap.2008.01.001Get rights and content

Abstract

Cyclophosphamide is one of the most often used anticancer drugs. Although DNA interstrand cross-links are considered responsible for its cytotoxicity, the mechanism of initiation and execution of cell death is largely unknown. Using the cyclophosphamide analogue mafosfamide, which does not need metabolic activation, we show that mafosfamide induces apoptosis dose and time dependently in lymphoblastoid cells, with clearly more apoptosis in p53wt cells. We identified two upstream processes that initiate apoptosis, DNA replication blockage and transcriptional inhibition. In lymphoblastoid cells, wherein DNA replication can be switched off by tetracycline, proliferation is required for inducing apoptosis at low dose mafosfamide. At high dose, transcriptional inhibition also contributes to cell death. The RNA synthesis inhibitor α-amanitin induced similar to mafosfamide more apoptosis in p53wt than in p53mt cells. In combination with mafosfamide, however, α-amanitin had no additive effect. Mafosfamide caused p53 stabilization by phosphorylation of Ser15, 20 and 37, and activation of ATM/ATR and Chk1/Chk2. Inhibition of ATM/ATR, PI3-kinase and Chk1/Chk2 by CGK733, wortmannin and DBH, respectively, attenuated the apoptotic response in p53wt but not p53mt cells. Mafosfamide induced caspase dependent apoptosis and, for low dose treated cells, caspases were preferentially activated in the S-phase, whereas at high dose caspases were activated in all cell cycle stages. These data support the conclusion that at low dose level of mafosfamide, DNA replication blockage is the dominant apoptosis-inducing event, while at high dose, transcriptional inhibition comes into play. The data provide a mechanistic explanation of why cyclophosphamide applied at therapeutic doses preferentially kills replicating tumor cells.

Introduction

Since its inception in 1958, cyclophosphamide has become one of the most frequently used anticancer drugs. It is used either as monotherapy or in combination with other drugs, e.g. with vincristine, doxorubicin and prednisone (CHOP schedule) (Younes, 2004) or with 5-fluorouracile and epirubicin (Collecchi et al., 1998), in the treatment of breast cancer, ovarian cancer, leukemia and lymphoma. Cyclophosphamide is also administered at low doses for immunosuppression of patients suffering from arthritic diseases (Vitali et al., 1993).

Cyclophosphamide belongs to the group of N-lost compounds. It requires CYP450-dependent activation, yielding the active compound 4-hydroxy-cyclophosphamide (Roy et al., 1999). This molecule undergoes spontaneous decomposition to the biologically active alkylating species phosphoramide mustard and acrolein (Mirkes, 1985). The former preferentially alkylates the N7 position of guanine. Since cyclophosphamide is a bifunctional compound, DNA alkylation gives rise to interstrand cross-links (ICLs) that are thought to be responsible for the induction of cell death (Erickson et al., 1980, Mirkes, 1985). The cyclophosphamide derivative mafosfamide does not require metabolic activation. Nevertheless, it acts in the same way as cyclophosphamide. It is only used for local treatment and in vitro studies (Pette et al., 1995, Blaney et al., 2005).

The cytotoxicity of cyclophoshamide and mafosfamide in vitro (Hemendinger and Bloom, 1996, Lee et al., 1997, Munker et al., 1998, Foot et al., 1999) and in vivo (Smeyne et al., 1994, Wang and Cai, 1999) was reported to be due to the induction of apoptosis. It is still unclear, however, whether the extrinsic (Lee et al., 1997, Wang and Cai, 1999) or the intrinsic (Foot et al., 1999, Venter et al., 2001, Stahnke et al., 2004) pathway dominates following treatment. Although caspases were shown to participate in the execution of N-lost mustard triggered apoptosis (Venter et al., 2001, Stahnke et al., 2004), there is still doubt about the involvement of caspases in cell death induced by these chemotherapeutic drugs (Stahnke et al., 2001).

Since the primary target of cyclophosphamide (and mafosfamide) is nuclear DNA, it is extremely important to elucidate the upstream DNA damage-activated pathways that trigger apoptosis. Furthermore, since the agents induce DNA cross-links, the possibility arises that transcriptional inhibition and/or DNA replication blockage are critical for cell death initiation. Transcriptional inhibition has been shown to induce apoptosis in α-amanitin (Schoepe et al., 1986, Ljungman et al., 1999, Arima et al., 2005) and actinomycin D treated cells (Dunkern and Kaina, 2002). Also, a correlation between transcriptional inhibition and the induction of p53 was demonstrated upon UV-C irradiation and cisplatin treatment (Ljungman et al., 1999). Therefore, we elucidated the role of transcription and replication inhibition in mafosfamide-induced cell death.

Several proteins recognize damaged DNA. ATM binds to DNA double-strand breaks (Gatei et al., 2001), phosphorylates Chk1, Chk2 and p53 and thus triggers cell cycle progression blockage DNA repair or apoptosis. ATR recognizes stalled replication forks and single-stranded DNA (Osborn et al., 2002) and, upon activation, phosphorylates Chk1 and p53. Therefore, it can also initiate a signaling cascade leading to apoptosis. Activation of ATR following UV irradiation was shown to occur proliferation dependently (Zhou et al., 2003, Sinha and Hader, 2002, Brown and Baltimore, 2003, Pommier et al., 2004) and independently (Saris et al., 1999). For UV light, it has been shown that apoptosis in fibroblasts strongly depends on the passage of cells through the S-phase, and it has been proposed that during S-phase bulky DNA lesions are transformed into DNA double-strand breaks (Dunkern and Kaina, 2002, Batista et al., 2007, Roos et al., 2007) that trigger apoptosis (Lips and Kaina, 2001). This is in accord with ATR phosphorylation of H2AX, which was stronger in the S-phase of the cell cycle following UV light treatment (Ward and Chen, 2001).

Cyclophosphamide is widely used in the therapy of lymphomas (Gisselbrecht and Mounier, 2006). Therefore, we utilized a lymphoblastoid cell system. We compared the apoptotic response to mafosfamide of the lymphoblastoid cell lines TK6 and WTK1 that are wild-type (p53wt) and mutated for p53 (p53mt), respectively. Our results show that mafosfamide is highly potent in triggering apoptosis in lymphoblastoid cells. Apoptosis occurs in both p53wt and p53mt cells. However, in p53wt cells mafosfamide was clearly more effective than in p53mt cells in triggering apoptosis. We also show that replicating lymphoblastoid cells are more vulnerable than non-replicating cells and that, depending on the dose applied, both DNA replication blockage and inhibition of transcription are critical in triggering apoptosis following mafosfamide treatment. Collectively, the results suggest that p53, Chk1 and Chk2 are predictive markers of lymphoma sensitivity to cyclophosphamide and cyclophosphamide-analogous drugs. The data obtained bear therapeutic implications that will also be discussed.

Section snippets

Cell culture and drug treatment

TK6 and WTK1 cells were a generous gift from Dr. K. Weber (Department of Radiation Therapy, University of Heidelberg, Germany). P493-6 cells were kindly provided by Prof. D. Eick (GSF, Munich, Germany) and described previously (Pajic et al., 2000). TK6 and WTK1 cells were cultivated in RPMI-1640, 0.4% penicillin/streptomycin, 107 μg/ml sodium pyruvate, 0.25 mg/l l-glutamine, 10% FCS, 0.6 g NaHCO3 at 37 °C in a humidified atmosphere at 93% air and 7% CO2. Cell culture of P493-6 cells is

Role of p53 in mafosfamide triggered apoptosis in lymphoblastoid cells

Firstly, we determined whether mafosfamide induces apoptosis in lymphoblastoid cells and whether p53 is involved. Quantifying the SubG1 population, the data show that mafosfamide triggers apoptosis in a dose (Fig. 1a) and time (Figs. 1b, c and d) dependent manner. TK6 cells are clearly more sensitive than WTK1 cells, which was true both for the low (2 μg/ml; Fig. 1b) and high (10 μg/ml; Fig. 1c) dose level.

To distinguish between apoptosis and necrosis, the Annexin V/PI double-staining method

Discussion

Cyclophosphamide is presumably the most frequently used anticancer drug, applied for the treatment of lymphomas and many solid tumors. It is also used in anti-inflammatory therapy and for the suppression of the immune response after organ transplantation. Although the clinical aspects of cyclophosphamide and its analogues are well understood, little is known about the mechanism underlying the induction of cell death. Since ICLs are formed in the DNA following treatment with these bifunctional

Acknowledgments

Work was supported by DFG Ka724 and SFB432. We thank Prof. Dr. D. Eick (GSF, München) for providing the P493-6 cell line.

References (57)

  • K. Hiramatsu et al.

    Monochloramine inhibits ultraviolet B-induced p53 activation and DNA repair response in human fibroblasts

    Biochim. Biophys. Acta

    (2006)
  • R. Munker et al.

    Further characterization of cyclophosphamide resistance: expression of CD95 and of bcl-2 in a CML cell line

    Leuk. Res.

    (1998)
  • A.J. Osborn et al.

    Checking on the fork: the DNA-replication stress-response pathway

    Trends Cell Biol.

    (2002)
  • M. Pette et al.

    Mafosfamide induces DNA fragmentation and apoptosis in human T-lymphocytes. A possible mechanism of its immunosuppressive action

    Immunopharmacology

    (1995)
  • W.P. Roos et al.

    DNA damage-induced cell death by apoptosis

    Trends Mol. Med.

    (2006)
  • D. Shechter et al.

    Regulation of DNA replication by ATR: signaling in response to DNA intermediates

    DNA Repair (Amst)

    (2004)
  • A. Solhaug et al.

    Role of cell signaling in B[a]P-induced apoptosis: characterization of unspecific effects of cell signaling inhibitors and apoptotic effects of B[a]P metabolites

    Chem. Biol. Interact

    (2005)
  • K. Stahnke et al.

    Activation of apoptosis pathways in peripheral blood lymphocytes by in vivo chemotherapy

    Blood

    (2001)
  • G.J. Wang et al.

    Relatively low-dose cyclophosphamide is likely to induce apoptotic cell death in rat thymus through Fas/Fas ligand pathway

    Mutat. Res.

    (1999)
  • I.M. Ward et al.

    Histone H2AX is phosphorylated in an ATR-dependent manner in response to replicational stress

    J. Biol. Chem.

    (2001)
  • A. Younes

    New treatment strategies for aggressive lymphoma

    Semin. Oncol.

    (2004)
  • S. Banin et al.

    Enhanced phosphorylation of p53 by ATM in response to DNA damage

    Science

    (1998)
  • L.F. Batista et al.

    Differential sensitivity of malignant glioma cells to methylating and chloroethylating anticancer drugs: p53 determines the switch by regulating xpc, ddb2, and DNA double-strand breaks

    Cancer Res.

    (2007)
  • S.M. Blaney et al.

    Intrathecal mafosfamide: a preclinical pharmacology and phase I trial

    J. Clin. Oncol.

    (2005)
  • M.N. Boddy et al.

    Replication checkpoint enforced by kinases Cds1 and Chk1

    Science

    (1998)
  • E.J. Brown et al.

    Essential and dispensable roles of ATR in cell cycle arrest and genome maintenance

    Genes Dev.

    (2003)
  • A.N. Davidoff et al.

    Cell-cycle disruptions and apoptosis induced by the cyclophosphamide derivative mafosfamide

    Exp. Hematol.

    (1993)
  • T.R. Dunkern et al.

    Cell proliferation and DNA breaks are involved in ultraviolet light-induced apoptosis in nucleotide excision repair-deficient Chinese hamster cells

    Mol. Biol. Cell

    (2002)
  • Cited by (0)

    View full text