Prolonged cell cycle arrest in irradiated human diploid skin fibroblasts: The role of nutrient deprivation

Ionizing radiation has been reported to cause an irreversible cell cycle ar rest in normal human diploid fibroblasts. However, colony survival assays s how that even at high doses of gamma radiation, human diploid fibroblasts d o not irreversibly arrest, and that a dose-dependent fraction is capable of continued cycling. In this study, we resolve the apparent discrepancy betw een colony survival assays and the observed radiation-induced prolonged arr est. Using how cytometry analysis, we have confirmed that human diploid fib roblasts do exhibit a prolonged cell cycle arrest in both G(1) and G(2)/M p hases of the cell cycle. However, a single replacement of fresh growth medi um stimulated a fraction of the arrested population of cells to transiently re-enter the cell cycle. Daily medium changes stimulated these irradiated human diploid fibroblasts to continue cycling until they were contact-inhib ited. Thus the fraction of human diploid fibroblasts which survive radiatio n exposure and are capable of cycling appears to permanently arrest as a re sult of nutrient insufficiency. Western blot analysis demonstrated a radiat ion-induced elevation in TP53 (formerly known as p53) protein levels within 2 h postirradiation, followed by a decrease to levels comparable to those in unirradiated controls. The TP53 and CDKN1A (formerly known as p21) prote in levels were indistinguishable after 24 h and remained elevated for a 6-d ay period of observation in both control and irradiated cultures. Our studi es indicate that human diploid fibroblasts are capable of re-entering the c ell cycle after exposure to ionizing radiation and that this re-entry is de pendent on a constant supply of nutrients provided by fresh medium changes. The fraction of cells capable of resuming cell cycling is consistent with the surviving fraction of cells in colony assays. (C) 2000 by Radiation Res earch Society.