Recovery of HeLa cell population growth after treatment with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB)

Abstract

Dose-response curves for the inhibition of heterogeneous nuclear RNA (hnRNA) synthesis in HeLa cells by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB; 5-100 microM; 30 min) are biphasic and indicate the existence of two subpopulations of hnRNA molecules, one highly sensitive and the other completely resistant, as previously reported for molecules greater than 1,000 nucleotides long (Tamm et al., 1976; Sehgal et al., 1976a). In the short-term experiments, the drug-sensitive synthesis of hnRNA was inhibited 50% at a DRB concentration of approximately 7 microM, and 70% at 20 microM, whereas drug-resistant synthesis, which comprises approximately 20% of total, continued at DRB concentrations as high as 100 microM. After 24 hr of DRB treatment in medium containing 5% fetal calf serum, the increase in cell number in the exponentially growing population was inhibited by only 42% at 20 microM DRB, and the formation of colonies of greater than or equal to ten cells was not decreased. DRB at 40 microM concentration decreased population growth by 76% and colony formation by 63%. Treatment with 60 microM DRB was sufficient to prevent a net increase in cell number and to reduce colony formation by 78%. After termination of treatment, the time required for the surviving population to begin rapid proliferation was directly related to the concentration of DRB used to treat cells and to the duration of treatment. After 24-hr treatment with 40 microM DRB, cultures recovered within 1 day, whereas after 60 microM DRB, 3-4 days were required. After 40-hr treatment with 60 microM DRB, 5-6 days were required for recovery, and after 80 microM DRB, 9-11 days. During the "dormant" period the cell number ranged from 15 to 60% of the initial number and was fairly stable for given conditions. After the "dormant" period, recovery was rapid. The population growth rate in cultures undergoing treatment with DRB is directly related to serum concentration; however, the recovery rate during the post-treatment period is unaffected by serum concentration.