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 arrest in normal human diploid fibroblasts. However, colony survival assays show that even at high doses of gamma radiation, human diploid fibroblasts do not irreversibly arrest, and that a dose-dependent fraction is capable of continued cycling. In this study, we resolve the apparent discrepancy between colony survival assays and the observed radiation-induced prolonged arrest. Using flow cytometry analysis, we have confirmed that human diploid fibroblasts do exhibit a prolonged cell cycle arrest in both G(1) and G(2)/M phases of the cell cycle. However, a single replacement of fresh growth medium 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-inhibited. Thus the fraction of human diploid fibroblasts which survive radiation exposure and are capable of cycling appears to permanently arrest as a result of nutrient insufficiency. Western blot analysis demonstrated a radiation-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) protein levels were indistinguishable after 24 h and remained elevated for a 6-day period of observation in both control and irradiated cultures. Our studies indicate that human diploid fibroblasts are capable of re-entering the cell cycle after exposure to ionizing radiation and that this re-entry is dependent 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.
DeSimone
J N
JN
Department of Microbiology and Molecular Genetics, University of California, Irvine, CA 92697, USA.
Dolezalova
H
H
Redpath
J L
JL
Stanbridge
E J
EJ
D015153:Blotting, Western; D002453:Cell Cycle / Q000528:radiation effects*; D018797:Cell Cycle Proteins; D002460:Cell Line; D002470:Cell Survival / Q000528:radiation effects; D003470:Culture Media; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D016213:Cyclins / Q000378:metabolism; D004171:Diploidy; D004307:Dose-Response Relationship, Radiation; D005347:Fibroblasts / Q000378:metabolism / Q000528:radiation effects; D005720:Gamma Rays; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D012867:Skin / Q000166:cytology / Q000378:metabolism / Q000528:radiation effects*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D025521:Tumor Suppressor Proteins
C493956:CDKN1A protein, human; D018797:Cell Cycle Proteins; D003470:Culture Media; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D008869:Microtubule-Associated Proteins; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27