title: Counteraction of pRb-dependent protection after extreme hypoxia by elevated ribonucleotide reductase.
abstract: We have studied hypoxia-induced cell cycle arrest in human cells where the retinoblastoma tumour suppressor protein (pRb) is either functional (T-47D and T-47DHU-res cells) or abrogated by expression of the HPV18 E7 oncoprotein (NHIK 3025 cells). We have previously found that pRb is dephosphorylated and rebound in the nucleus in T-47D cells arrested in S-phase during hypoxia and that this binding is protracted even following re-oxygenation. In the present study, however, we show that the long-lasting arrest following re-oxygenation induced by pRb-binding in the cell nuclei may be overruled by an elevated level of ribonucleotide reductase (RNR). This seems to create a forced DNA-synthesis, uncoordinated with cell division, which induces endoreduplication of the DNA. The data indicate that the cells initiating endoreduplication continue DNA-synthesis until all DNA is replicated once and then may start cycling and cell division with a doubled DNA-content. Corresponding data on the pRb-incompetent NHIK 3025-cells show similar endoreduplication in these. Thus, the data indicate that endoreduplication of DNA following re-oxygenation may come, either as a result of hypoxic arrest of DNA-synthesis when pRb-function is absent in the cells, or if it is overruled by increased RNR. The present study further shows that pRb not only protects the culture by arresting most of the cells that are exposed to extreme hypoxia in S-phase, but also increases cell survival by means of increased clonogenic ability of these cells. Interestingly, however, cells having an elevated level of RNR have equally high survival as wild-type cells following 20 h extreme hypoxia. If RNR-overruling of pRb-mediated arrest following re-oxygenation results in an unstable genome, this may therefore represent a danger of oncogenic selection as the protective effect of pRb on cell survival seems to be maintained.
authors.affiliation: Department of Physics, The Biophysics Group, The University of Oslo, Oslo, Norway. pal.graff@fys.uio.no
authors.forename: P
authors.initials: P
authors.lastname: Graff
authors.forename: J
authors.initials: J
authors.lastname: Seim
authors.forename: Ø
authors.initials: Ø
authors.lastname: Amellem
authors.forename: H
authors.initials: H
authors.lastname: Arakawa
authors.forename: Y
authors.initials: Y
authors.lastname: Nakamura
authors.forename: K K
authors.initials: KK
authors.lastname: Andersson
authors.forename: T
authors.initials: T
authors.lastname: Stokke
authors.forename: E O
authors.initials: EO
authors.lastname: Pettersen
mesh_terms: D002453:Cell Cycle / Q000235:genetics*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D002455:Cell Division / Q000235:genetics; D015687:Cell Hypoxia / Q000235:genetics; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000235:genetics; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D004261:DNA Replication / Q000235:genetics; D006801:Humans; D009857:Oncogenes / Q000235:genetics; D010100:Oxygen / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D016160:Retinoblastoma Protein / Q000235:genetics / Q000378:metabolism*; D012264:Ribonucleotide Reductases / Q000378:metabolism*; D016196:S Phase / Q000235:genetics; D014410:Tumor Stem Cell Assay; D015854:Up-Regulation / Q000235:genetics*
chemical_list: D018797:Cell Cycle Proteins; D016160:Retinoblastoma Protein; D012264:Ribonucleotide Reductases; D010100:Oxygen