Abstract
ZD2767P is a phenol mustard glutamate prodrug which is currently being developed for Antibody Directed Enzyme Prodrug Therapy (ADEPT). In ZD2767 ADEPT an active bi-functional alkylating drug, ZD2767D (4-[N,N-bis(2-iodoethyl)amino]phenol), is generated following cleavage of ZD2767P by the bacterial enzyme carboxypeptidase G2 (CPG2) which is targeted to the tumour by conjugation to the F(ab′)2fragment of the anti-CEA antibody A5B7. The aim of the studies described here was to identify the mode of cell death induced by ZD2767P + CPG2 in comparison to the established nitrogen mustard chlorambucil. The contribution of bifunctional and monofunctional ZD2767 DNA lesions to cell death induction was investigated using a monofunctional ZD2767D analogue. Apoptosis in LoVo tumour cells was studied by three different methods (nuclear morphology, annexin V staining and TUNEL). Levels of apoptosis detected using the three assays were similar, and each drug treatment produced apoptosis at levels above those in control cells at concentrations which resulted in tumour cell growth inhibition. The bi-functional compounds, ZD2767P + CPG2 and chlorambucil, induced apoptosis in a concentration and time dependent manner, with equitoxic concentrations producing equivalent levels of apoptosis. In contrast, the mono-functional ZD2767D analogue at 100 μM resulted in the maximal level of apoptosis at 25 h with no further increase over the following 72 h. These studies have demonstrated that apoptosis is the mechanism of cell death induced by the ZD2767 ADEPT system, and that levels of apoptosis produced by ZD2767 are similar to those observed at equitoxic concentrations of the classical nitrogen mustard chlorambucil. The mono-functional ZD2767 analogue also induced apoptosis, but with a different time course and characteristics. In conjunction with previous data, these studies have shown that the potent activity of ZD2767 can be attributed to the ability of the compound to induce bifunctional DNA lesions and engage apoptosis. © 2001 Cancer Research Campaign www.bjcancer.com
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References
Bagshawe, KD (1987). Antibody directed enzymes revive anti-cancer prodrugs concept. Br J Cancer, 56, 531–532.
Begleiter, A, Lee, K, Israels, LG, Mowat, MRA & Johnston, JB (1994). Chlorambucil induced apoptosis in chronic lymphocytic leukemia (CLL) and it’s relationship to clinical efficacy. Leukemia, 8, S103–S106.
Blakey, DC, Burke, PJ, Davies, DH, Dowell, RI, East, SJ, Eckersley, KP, Fitton, JE, McDaid, J, Melton, RG, Niculescu-Duvaz, IA, Pinder, PE, Sharma, SK, Wright, AF & Springer, CJ (1996). ZD2767, an improved system for antibody-directed enzyme prodrug therapy that results in tumour regressions in colorectal tumour xenografts. Cancer Res, 56, 3287–3292.
Chen, T (1977). In Situ detection of mycoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain. Exp Cell Res, 104, 255–262.
Fadok, VA, Voelker, DR, Campbell, PA, Cohen, JJ, Bratton, DL & Henson, PM (1992). Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J Immunol, 148, 2207–2216.
Fernandes, RS & Cotter, TG (1994). Apoptosis or necrosis: Intracellular levels of glutathione influence mode of cell death. Biochem Pharmacol, 48, 675–681.
Gavrieli, Y, Sherman, Y & Ben-Sasson, SA (1992). Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol, 119, 493–501.
Guchelaar, H-J, Vermes, I, Koopmans, RP, Reutelingsperger, CPM & Haanen, C (1998). Apoptosis-and necrosis-inducing potential of cladribine, cytarabine, cisplatin and 5-fluorouracil in vitro: a quantitative pharmacodynamic model. Cancer Chemother Pharmacol, 42, 77–83.
Hickman, JA & Boyle, CC (1997). Apoptosis and Cytotoxins. Br Med Bull, 52, 632–643.
Lennon, SV, Martin, SJ & Cotter, TG (1991). Dose-dependent induction of apoptosis in human tumour cell lines by widely diverging stimuli. Cell Prolif, 24, 203–214.
Lootiens FG Regenfuss, P, Zechel, A, Dumortier, L & Clegg, RM (1990). Binding characteristics of Hoechst 33258 with calf thymus DNA, poly[d(A-T)], and d(CCGGAATTCCGG): Multiple stoichiometries and determination of tight binding with a wide spectrum of site affinities. Biochemistry, 29, 9029–9039.
Martin, SJ, Reutelingsperger, CPM, McGahon, AJ, Rader, JA, van Schie, RCAA, LaFace, DM & Green, DR (1995). Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: Inhibition by overexpression of Bcl-2 and Abl. J Exp Med, 182, 1545–1556.
Pepper, C, Hoy, T & Bentley, P (1997). Bcl-2/Bax ratios in chronic lymphocytic leukaemia and their correlation with in vitro apoptosis and clinical resistance. Br J Cancer, 76, 935–938.
Pepper, C, Hoy, T & Bentley, P (1998a). Elevated Bcl-2/Bax are a consistent feature of apoptosis resistance in Bcl-2 chronic lymphocytic leukaemia and are correlated with in vivo chemoresistance. Leukemia and Lymphoma, 28, 355–360.
Pepper, C, Thomas, A, Tucker, H, Hoy, T & Bentley, P (1998b). Flow cytometric assessment of three different methods for the measurement of in vitro apoptosis. Leuk Res, 22, 439–444.
Roberts, JJ, Brent, TP & Crathorn, AR (1971). Evidence for the inactivation and repair of the mammalian DNA template after alkylation by mustard gas and half mustard gas. Eur J Cancer, 7, 515–524.
Sgonc, R, Boeck, G, Dietrich, H, Gruber, J, Recheis, H & Wick, G (1994). Simultaneous determination of cell surface antigens and apoptosis. Trends Genet, 10, 41–42.
Sherwood, RF, Melton, RG, Alwan, SM & Hughes, P (1985). Purification and properties of carboxypeptidase G2 from Pseudomonas sp. strain RS-16. Use of a novel triazine dye affinity method. Eur J Biochem, 148, 447–453.
Springer, CJ, Dowell, R, Burke, PJ, Hadley, E, Davies, DH, Blakey, DC, Melton, RG & Niculescu-Duvaz, I (1995). Optimization of alkylating agent prodrugs derived from phenol and aniline mustards: A new clinical candidateprodrug (ZD2767) for antibody-directed enzyme prodrug therapy (ADEPT). J Med Chem, 38, 5051–5065.
Umansky, SR (1996). Apoptosis: Molecular and cellular mechanisms (A review). Mol Biol, 30, 285–295.
Vermes, I, Haanen, C, Steffens-Nakken, H & Reutelingsperger, C (1995). A novel assay for apoptosis flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled annexin V. J Immunol Methods, 184, 39–51.
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Monks, N., Blakey, D., Curtin, N. et al. Induction of apoptosis by the ADEPT agent ZD2767: comparison with the classical nitrogen mustard chlorambucil and a monofunctional ZD2767 analogue. Br J Cancer 85, 764–771 (2001). https://doi.org/10.1054/bjoc.2001.1947
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DOI: https://doi.org/10.1054/bjoc.2001.1947
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