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Biotech / Medical : Millennium Pharmaceuticals, Inc. (MLNM) -- Ignore unavailable to you. Want to Upgrade?

To: Icebrg who wrote (1697)	7/3/2003 12:35:58 PM
From: Icebrg	Read Replies (1) \| Respond to of 3044

Comparison of the cellular exposure time of XR5944 (MLN944) and known topoisomerase inhibitors required to induce cell cycle arrest, apoptosis and ultimate cell death. [Posted by PGS to the Yahoo board] Caroline Freathy, Wendy Dangerfield, Darshan S. Sappal, Steven G. Kovats, Prakash Mistry, Laura A. Rudolph-Owen, Peter Charlton. Xenova Ltd, Slough, UK; Millennium Pharmaceuticals, Inc, Cambridge, MA. The bis-phenazine, XR5944 is a novel agent with potent activity against both human and murine tumour cells (Anti-Cancer Drugs 2001, 12:359). The current study examined the effect of exposure time on the ability of XR5944 to induce adverse cellular events and ultimate cell death on human carcinoma cell lines. HCT116 and HT29 colon carcinoma cells were plated and subsequently treated with XR5944, doxorubicin or SN38 (the active metabolite of irinotecan) for 30 minutes to 96 hours. Aberrations in the cell cycle were assessed by flow cytometry and cell survival assessed colorimetrically with the WST assay. Synchronized cells exposed to 20 nM XR5944 (EC80) for one hour exhibit cell cycle aberrations. These observed cell cycle changes include arrest in the G1 and G2 phases of the cell cycle in addition to a reduction of cells in the S phase of the cell cycle. Standard topoisomerase inhibitors such as doxorubicin and camptothecin (CPT) exhibited increased cytotoxic potency (decrease in EC50) with an increase in exposure time. In the HCT116 and HT29 cells potency of both doxorubicin and SN38 increased by ³235-fold with an increase in exposure time from 30 min to 48 (HCT116) or 96 (HT29) hours. In contrast the high potency of XR5944 did not appear to be dependent on exposure time and only increased by 2 to 4-fold with an increase in exposure time from 30 min to 96 hours. At 48 hours the EC50for XR5944 in the HCT116 and HT29 cells was 0.5 and 36 nM. Exposure to XR5944 appears to result in a time and concentration-dependent increase in apoptosis in L23/P(NSCLC) and HT29 cells as assessed by the exposure of phosphatidyl serine residues on the cell membrane. The percentage of apoptotic cells reached a maximum of 30-40% after 24 hours in the L23/P cell line and 48 hours in the HT29 cell line with concentrations in the 200-400nM range. This level of apoptosis was comparable to that seen with CPT and etoposide, well-documented inducers of apoptotic cell death (Biochim Biophys Acta 1998, 400:195). In addition, exposure to the same concentrations of XR5944 appeared to cause a significant increase in caspase-3/7 activity, the major effector caspases primarily responsible for demolition of the cell. However, the maximum caspase activity observed with XR5944 was consistently 50-60% lower than that seen with the positive controls, suggesting that effector caspases other than caspase-3 and 7 may play an important role in XR5944-induced apoptosis. Conclusions: XR5944 is a potent new cytotoxic that appears to be capable of inducing cellular toxicity within an extremely short time period compared to control topoisomerase inhibitors. Classical hallmarks of apoptosis, however, appeared within the same time course as standard topo inhibitors that are known to induce apoptotic cell death. These data suggest the mechanism of action of XR5944 is distinct from standard topo I and topo II directed agents. aacr03.agora.com