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

Brain Tumor Research Center, Department of Neurological Surgery [R. J., D. F. D.], and Department of Radiation Oncology [D. F. D.], School of Medicine, University of California, San Francisco, San Francisco, California 94143

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 population was estimated using a fluorometric method. Cross-link formation in the noncycling spheroid cells was calculated from these DNA measurements and the percentage of cycling cells as determined by autoradiography. The isotopic and fluorometric assays yielded equivalent elution profiles in cells irradiated with an X-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.

¹ Supported by USPHS Grants CA 31868 and CA 13525 and by the Aaron Silvera Fund.

² To whom requests for reprints should be addressed at The Brain Tumor Research Center (783-HSW), Department of Neurological Surgery, School of Medicine, University of California, San Francisco, San Francisco, CA 94143.

Received 5/13/85. Revised 11/27/85. Accepted 1/29/86.