Abstract
P450 reductase (NADPH: cytochrome c (P450) reductase, EC 1.6.2.4) plays an important role in the reductive activation of the bioreductive drug tirapazamine (SR4233). Thus, in a panel of human breast cancer cell lines, expression of P450 reductase correlated with both the hypoxic toxicity and the metabolism of tirapazamine [Patterson et al (1995) Br J Cancer 72: 1144-1150]. To examine this dependence in more detail, the MDA231 cell line, which has the lowest activity of P450 reductase in our breast cell line panel, was transfected with the human P450 reductase cDNA. Isolated clones expressed a 78-kDa protein, which was detected with anti-P450 reductase antibody, and were shown to have up to a 53-fold increase in activity of the enzyme. Using six stable transfected clones covering the 53-fold range of activity of P450 reductase, it was shown that the enzyme activity correlated directly with both hypoxic and aerobic toxicity of tirapazamine, and metabolism of the drug under hypoxic conditions. No metabolism was detected under aerobic conditions. For RSU1069, toxicity was also correlated with P450 reductase activity, but only under hypoxic conditions. Measurable activity of P450 reductase was found in a selection of 14 primary human breast tumours. Activity covered an 18-fold range, which was generally higher than that seen in cell lines but within the range of activity measured in the transfected clones. These results suggest that if breast tumours have significant areas of low oxygen tension, then they are likely to be highly sensitive to the cytotoxic action of tirapazamine and RSU 1069.
Highlights
Western blot analysis using a polyclonal antibody raised against recombinant P450 reductase revealed a single band with an apparent molecular weight of 78 kDa, which co-migrated with endogenous P450 reductase
We have successfully transfected into the human breast cancer MDA231 cell line a DNA construct encoding the gene for the human form of P450 reductase
Under hypoxic conditions, P450 reductase was strongly implicated in activation and subsequent toxicity, whereas, in air, expression of reductase was only important for the toxicity of tirapazamine
Summary
Tirapazamine, SR 4317, SR 4330 and RSU 1069 were synthesized in house using previously described methods (Seng and Ley, 1972; Adams et al, 1984). NADPH was purchased from Boehringer Mannheim (Lewes, UK), HPLC grade methanol was purchased from Merck (Lutterworth, UK). All other reagents were of analytical grade and were purchased from Sigma (Poole, UK). Tissue culture media was obtained from ICRF (Clare Hall Labs, UK) and fetal calf serum from Sigma. Growth conditions for the six human breast tumour cell lines used in this work have been described previously (Houlbrook et al, 1994; Patterson et al, 1995). All cell lines were maintained in exponential growth phase in RPMI 1640 medium (except for SKBr-3 cells, which were maintained in Dulbecco's modified Eagle medium), supplemented with 2 mm glutamine and 10% (v/v) fetal calf serum
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
