Differential Drug Sensitivity Conferred by Growth Status Detected in a Mixed Population of Cycling and Noncycling Cells1

The noncycling cell compartment of tumors is considered to be an important target for chemotherapeutic agents; yet, it has been difficult to accurately quantitate its contribution to tumor response because of a lack of methods that can readily discern the relative sensitivities of cycling and noncycling cells. We have used antibodies against bromodeoxyuridine-substituted DNA in a unique experimental protocol that provides a basis for distinguishing the cycling and noncycling cell compartments and detecting their respective levels of drug-induced chromosome damage. A mixed population of cycling and noncycling cells was obtained by culturing 9L rat brain tumor cells as multicellular spheroids. Cell cycle compart ments and phase distributions were monitored with flow cytometry using bivariate analysis of DNA content and bromodeoxyuridine incorporation. Bromodeoxyuridine labeling was manipulated to differentially label metaphases from cycling and noncycling cells for sister chromatid exchange. This is based on the differential staining patterns of chromatids and metaphases that are obtained from cells that have replicated in the presence of bromodeoxyuridine. The chromosome damage in each cell cycle compartment following exposure to the chemotherapeutic agent l,3-bis(2-chloroethyl)-l-nitrosourea was assessed by the number of sister chromatid exchanges induced by treatment. Noncycling cells were shown to be more sensitive to l,3-bis(2-chloroethyl)-l-nitrosourea-induced dam age than were cycling cells. However, when allowed to remain noncycling for 24 h after treatment, the number of exchanges decreased in noncycling cells, which may indicate their ability to recover from damage. These results illustrate an experimental protocol that permits simultaneous assessment of cell cycle compartment recruitment and sister chromatid exchange induction in cells derived from a cytokinetically complex pop ulation containing both cycling and noncycling cells.