Double Elution Analysis of Cross-Link Formation and Repair in Cycling and Noncycling 9L Spheroid Cells1

We developed the double elution analysis technique, a modification of standard alkaline elution techniques, to assess cross-link formation and repair in the cycling and noncycling cell populations of multicellular spheroids. DNA cross-linking from cycling cells was measured using radioisotope incorporation; DNA cross-linking from the total cell popu lation was estimated using a fluorometric method. Cross-link formation in the noncycling spheroid cells was calculated from these DNA mea surements and the percentage of cycling cells as determined by autoradiography. The isotopie and fluorometric assays yielded equivalent elu tion profiles in cells irradiated with an \-ray dose of 6 Gy, and DNA interstrand cross-link formation by nitrogen mustard was equivalent in the cycling and the total cell population. The rate of cross-link removal appeared to be faster, however, in the cycling cell population. Double elution analysis should be applicable for the investigation of a variety of antineoplastic drugs that produce cross-links and for measuring damage to cycling and noncyling cell populations in tumors from experimental animals, as well as in multicellular spheroid tumor models.