Comments on 'Radiation-Induced DNA Unwinding Is Influenced by Cell Shape and Trypsin'

We have read the paper by Olive and MacPhail (1) with great interest. Their data indicate that: (a) "trypsin did not influence cell killing by ionizing radiation" (i.e., the radiation sensitivity of round cells in spheroids with and without trypsin treatment was the same), and (b) the increase in the radiation resistance and the limited DNA unwinding ability (as measured by the alkaline unwinding assay) of V79 spheroid cells appear to be contingent upon a change in cell shape. Their results and their interpretation of our data are pertinent to the observations on the relationship between radiosensitivity, trypsin, cell shape, and chromatin structure published by us for V79 (S-171) cells (2-5). Our data show that: (a) trypsin can radiosensitize spread monolayer cells (2-5); (b) trypsin cannot radiosensitize round mitotic cells (2), round cells in spheroids (4), or round mouse lymphoma cells (L5178Y-S) in suspension (5); (c) radiosensitization by trypsin is related to cell shapedependent chromatin structure (2-5); and (d) the degree of unwinding ofchromatin in round cells is higher than that of spread monolayer cells (5). There has also been sporadic mention of a possible radiosensitizing effect by trypsin [(6, 7), and see Ref. (2) for additional references on trypsin]. The observation by Olive and MacPhail (1) that trypsin did not radiosensitize round cells in spheroids is essentially the same as our results for round mitotic cells (2) or round cells in spheroids (4) or for round L5178Y-S cells (5). However, their other observations that spheroid cells were more radioresistant than monolayer cells and that the degree of DNA unwinding in the former cells was lower than that of the latter cells are not in agreement with our results. Our results showed that the radiosensitivity and the degree of DNA unwinding (as measured by the nucleoid halo technique) of monolayer and spheroid cells treated with trypsin was nearly the same (5). The reason for this difference in results is not clear. Lest any confusion arise from their misquotation of our observations (2-5) in their paper (1) pertaining to the effects of trypsin on radiosensitivity, we note the following: In the last sentence of the first paragraph of the Discussion, they state: "Recent results suggest that treatment of V79-171S cells with trypsin increases their resistance to radiation damage, an effect which appears to be related to differences in cell shape between monolayers and spheroids (18)". [Their Ref. (18) is Ref. (4) here]. What we have repeatedly shown is that: (a) spread cells in monolayers treated with trypsin either immediately before or immediately after irradiation are more radiosensitive than those cells which have been treated with trypsin 2-3 h after irradiation and repair incubation, and (b) the radiosensitization under immediate plating conditions is correlated with the rounding of spread cells by trypsin (2-5). Reference to mouse lymphoma cells (L5178Y) as monolayers in Table I appears to be a typographical error.