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DISCUSSION PAPER: CYTOTOXIC MECHANISMS OF FOLATE ANTAGONISTS

In this discussion I should like to comment on three separate points. The first will deal with some of the differences observed by different investigators with regard to the characteristics of cell kill by amethopterin (Methotrexate) (MTX). The second point is brief and is related to the selection of new folic acid analogues which, in some mammalian cell systems, may be superior to MTX as cell killing agents. The third point will deal, again briefly, with one aspect of the problem of predicting the clinical outcome of treatment. The area relating to the first point is best defined by comparing the findings of Dr. Hryniuk and associates on the L5178Y system as presented earlier in this volume and in the literature 1 with the published results of Borsa and Whitmore,a who worked with an L-cell system. In the L5178Y system cell killing begins immediately upon addition of MTX, and there is more cell kill during a four-hour exposure in the absence of exogenous purine than in its presence. In the L-cell system, cell killing begins only after a delay of approximately 10 hours, and the rate of cell killing is much greater in the presence of exogenous purine than in its absence. The delayed onset type of MTX-induced cell killing is also operative in the Hela cell system of Rueckert and Mueller.:’ Thus in the L5178Y system, cell kill is very rapid and is diminished in the presence of exogenous purine, whereas in the L-cell system and in the Hela cell system the cell kill is of a delayed type and in L-cells at least, is greatly augmented in the presence of exogenous purine. Two possibilities exist to explain these two types of cell kill characteristics. One is that there is a basic difference in killing mechanism operative in the different cell systems employed. The other possibility is that the differences are due to procedural artifacts. One possible source of such variation stems from the different conditions under which cells can be assayed for viability following drug treatment. In our work with L-cells, we assayed for cells which, following treatment with MTX, could grow into colonies in medium that contained both thymidine (TdR) and purines. Thus the MTX-induced thymineless and/or purineless state was abruptly brought to an end at the time of plating of the cells. Our reasoning in this was that the assay condition for viability should simulate the worst possible condition in vivo where TdR and purines might be made available to treated cells from degradation of other cells, perhaps previously killed by the MTX treatment. Rueckert and Mueller also included TdR in their plating medium. Under this assay condition, the cell kill was of the delayed type as previously mentioned. One might get different results if the assay for viable cells were carried out in medium lacking TdR and purines, as it was in Dr. Hryniuk’s L5178Y system. In such a case the MTX-induced thymineless and/or