• Hypoxic human cancer cells are sensitized to BH-3 mimetic–induced apoptosis via downregulation of the Bcl-2 protein Mcl-1.

      Harrison, Luke R; Micha, Dimitra; Brandenburg, Martin; Simpson, Kathryn L; Morrow, Christopher J; Denneny, Olive; Hodgkinson, Cassandra L; Yunus, Zaira; Dempsey, Clare E; Roberts, Darren L; et al. (2011-03-01)
      Solid tumors contain hypoxic regions in which cancer cells are often resistant to chemotherapy-induced apoptotic cell death. Therapeutic strategies that specifically target hypoxic cells and promote apoptosis are particularly appealing, as few normal tissues experience hypoxia. We have found that the compound ABT-737, a Bcl-2 homology domain 3 (BH-3) mimetic, promotes apoptotic cell death in human colorectal carcinoma and small cell lung cancer cell lines exposed to hypoxia. This hypoxic induction of apoptosis was mediated through downregulation of myeloid cell leukemia sequence 1 (Mcl-1), a Bcl-2 family protein that serves as a biomarker for ABT-737 resistance. Downregulation of Mcl-1 in hypoxia was independent of hypoxia-inducible factor 1 (HIF-1) activity and was consistent with decreased global protein translation. In addition, ABT-737 induced apoptosis deep within tumor spheroids, consistent with an optimal hypoxic oxygen tension being necessary to promote ABT-737–induced cell death. Tumor xenografts in ABT-737–treated mice also displayed significantly more apoptotic cells within hypoxic regions relative to normoxic regions. Synergies between ABT-737 and other cytotoxic drugs were maintained in hypoxia, suggesting that this drug may be useful in combination with chemotherapeutic agents. Taken together, these findings suggest that Mcl-1–sparing BH-3 mimetics may induce apoptosis in hypoxic tumor cells that are resistant to other chemotherapeutic agents and may have a role in combinatorial chemotherapeutic regimens for treatment of solid tumors.
    • Optimisation of circulating biomarkers of cell death for routine clinical use.

      Greystoke, Alastair; Cummings, Jeffrey; Ward, Timothy H; Simpson, Kathryn L; Renehan, Andrew G; Butt, Fouziah; Moore, David; Gietema, J; Blackhall, Fiona H; Ranson, Malcolm R; et al. (2008-05)
      BACKGROUND: M30 and M65 enzyme-linked immunosorbent assays detect circulating cytokeratin 18 fragments released during caspase-dependent or total cell death, respectively, and have potential as biomarkers in epithelial cancers. While these assays have been validated, their robustness for routine clinical use is unknown. PATIENTS AND METHODS: M30 and M65 were measured in matched serum and plasma samples from 31 lung cancer patients and 18 controls. RESULTS: Time allowable between sample acquisition and processing is critical for assays in clinical use. A 4-h delay in processing at room temperature increased M30 (P < 0.0001), an effect minimised by incubation on ice. M30 and M65 in serum were resistant to processing variations including delays. Serum and plasma measurements correlated well although M30 but not M65 was lower in serum (P < 0.0005). Less variation between duplicate assays was observed in serum. Prolonged storage (-80 degrees C) led to increased M30 (12%, 6 months; 34%, 1 year). Sample dilution in the supplied assay diluent proved non-linear, whereas dilution in donor serum or porcine plasma restored linearity up to a ratio of 1 : 6. CONCLUSION: We present recommendations that improve the reliability of these assays for clinical use and recommend serum as the preferred matrix with data more resistant to variations in collection.