Deficiencies in DNA replication and cell-cycle progression in polyamine-depleted HeLa cells.
Synchronized HeLa cells depleted of polyamines by alpha-difluoromethylornithine exhibited substantially decreased DNA synthesis, and proliferation ceased after the release of the cells into S phase. Nuclei from these cells synthesized 70-80% less DNA than did nuclei from control cells. Extraction of isolated nuclei with 0.3 M-KCl decreased DNA synthesis by about 60%, which was recovered almost completely in control cell nuclei by reconstitution with the salt extracts of these nuclei. On the other hand, salt extracts of polyamine-depleted nuclei restored only 50% of DNA synthesis in extracted control nuclei. Salt extracts of control cell nuclei contained twice the DNA polymerase alpha activity of polyamine-depleted nuclear extracts. Extracts of cell lysates of both control and polyamine-depleted HeLa cells exhibited similar DNA polymerase alpha activity, suggesting that uptake of the enzyme or its retention by the nuclei of polyamine-depleted cells was decreased. Polyamine-depleted nuclei also showed altered phosphorylation of a 31 kDa protein as compared with control nuclei. Almost normal DNA synthesis, cell proliferation, DNA polymerase alpha activity and nuclear protein phosphorylation were restored in polyamine-depleted cells grown in medium supplemented with 20 microM-spermidine at least 10-12 h before S phase. Cultures in which proliferation was blocked by alpha-difluoromethylornithine did not exhibit synchronous growth after the block was removed. Thus it may be concluded that HeLa cells depleted of polyamines are not inhibited at a single control point in the cell cycle, but are arrested at diverse sites throughout G1 phase.
Koza
R A
RA
Department of Biochemistry, University of New Hampshire, Durham, NH 03824.
Herbst
E J
EJ
D002453:Cell Cycle / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002467:Cell Nucleus / Q000187:drug effects / Q000502:physiology; D004257:DNA Polymerase II / Q000378:metabolism*; D004261:DNA Replication / Q000187:drug effects; D000518:Eflornithine / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism; D011073:Polyamines / Q000378:metabolism*; D011700:Putrescine / Q000378:metabolism; D016196:S Phase; D013095:Spermidine / Q000378:metabolism / Q000494:pharmacology; D013096:Spermine / Q000378:metabolism; D013938:Thymidine Monophosphate / Q000378:metabolism; D014316:Tritium
D009687:Nuclear Proteins; D011073:Polyamines; D014316:Tritium; D013096:Spermine; D013938:Thymidine Monophosphate; D004257:DNA Polymerase II; D013095:Spermidine; D011700:Putrescine; D000518:Eflornithine