The Effect of Alkylating Agents on Tumour Cell ATPase Enzymes

Proposals for the mechanism of action of antitumour nitrogen mustards are reviewed including those which suggest that targets other than DNA may be important for cytotoxicity. The cell membrane as a target for the cytotoxic action of antitumour agents is reviewed. The properties of the cell membrane enzyme sodium potassium dependent magnesium adenosine triphosphatase (Na⁺K⁺ATPase),which is considered to be synonymous with the "sodium pump", are described and the role of the enzyme and that of particular ions (Na⁺,K⁺,Mg²⁺ and Ca⁺) in cellular metabolism and the regulation of cell replication is reviewed.

The characterisation of enzymes in a crude cell] membrane preparation from a plasmacytoma cell line (ADJ/PC6) sensitive to nitrogen mustards in vivo, which utilise adenosine triphosphate as a substrate, is described and their activity is compared with those from other sources investigated.  Na⁺K⁺ATPase, magnesium ATPase, p-nitrophenolphosphatase and external Ca²⁺ ATPase activities are described. Nitrogen mustard (bis (B-chloroethyl) methylamine) and other alkylating agents are shown to be primarily inhibitors of Na⁺K⁺ATPase activity of the crude cell membrane preparation. Nitrogen mustard inhibits the enzyme in a time dependent manner with 100% inhibition at 10^-10M after 30 minutes. A monofunctional analogue of nitrogen mustard (N-N-dimethylchloroethy] amine) which has no antitumour activity was less potent: 100% inhibition at 10^-5M after 30 minutes. The uptake of rubidium (Rb⁺) into intact PC6 cells was measured and shown to be ouabain sensitive which suggests it is via the sodium pump.  Nitrogen mustard is shown to inhibit Rb⁺ uptake in a time dependent manner: 10^-5M reduced uptake by 45% after 4 hours.
However, 10^-6M nitrogen mustard, a concentration shown to kill 90% of PC6 cells in an in vitro - in vivo assay, had no effect on Rb⁺ uptake even after 6 hours.

The results are discussed with regard to a hypothesis that inhibition of tumour cell Na⁺ K⁺ ATPase nitrogen mustard
may be a cytotoxic lesion.