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Evaluation of Growth Fractions with Monoclonal Antibodies to Human -DNA Polymerase¹

Department of Pharmacology [A. A., A. N.] and Clinical Oncology [P. F. C.], Instituto Nazionale Ricerca Cancro, Genova, Italy 16132, and Division of Laboratory Medicine [B. D.], University of Texas, M. D. Anderson Hospital & Tumor Institute at Houston, Houston, Texas 77030

We have established and partially characterized a panel of monoclonal antibodies against -DNA polymerase. One of the hybridomas, clone 5F, has been exploited for cell kinetic studies on three colon cancer cell lines, LOVO, SW 620, and SW 403, which are endowed with different growth patterns and differentiation status. By an immunoperoxidase method, we could demonstrate the specific intranuclear localization of -DNA polymerase during the exponential phase of in vitro growth and contrast it with the diffuse distribution of the enzyme throughout the cytoplasm during the resting state. The percentage of intranuclear staining positive cells, evaluated at successive time points of in vitro growth, changed from 75 to 95% (assayed on Days 3 and 7) to 15 to 25% in confluent and resting populations assayed on Days 12 to 14. In agreement with the assumption that the enzyme moves from nucleus to cytoplasm after entering quiescence, -DNA polymerase was still present in the cytoplasm or in the cytoplasmic perinuclear area of cells in resting phase cultures. Comparisons between traditional kinetic parameters (thymidine labeling index and primer-dependent -DNA polymerase) and proliferative state determined by the monoclonal antibody supported the feasibility of this approach to define the proportion of actively proliferating elements in a tumor cell population. Moreover, parallel flow cytometric analysis performed on Days 5 and 14 of continuous culture showed fluctuations of -DNA polymerase content in relation to exponential and steady-state phases, with a significant increase in the amount of -DNA polymerase in actively proliferating populations and a progressive reduction of the enzyme as the cultures entered the resting stage.

¹ Supported in part by Grant CA23272 from the National Cancer Institute and by CNR Progetto Finalizzato Oncologia Grant 85.02108.44.

² To whom requests for reprints should be addressed, at Department of Pharmacology, Istituto Nazionale Ricerca Cancro, Viale Benedetto XV, 10, 16132 Genova, Italy.

Received 6/ 9/86. Revised 11/ 3/86. Accepted 12/12/86.