Date of Award
Program or Major
Doctor of Philosophy
Edward J Herbst
HeLa cells were synchronized for S-phase DNA synthesis and simultaneously depleted of polyamines by the addition of the inhibitor of polyamine biosynthesis, $\alpha$-difluoromethylornithine (DFMO).
DFMO reacts catalytically with and irreversibly inhibits the enzyme ornithine decarboxylase (ODC) thereby preventing the biosynthesis of the polyamines putrescine, spermidine, and spermine.
The depletion of polyamines in HeLa cells inhibits cellular processes such as DNA synthesis and proliferation. These processes could be reversed by the addition of spermidine 10-12 hours before S-phase DNA synthesis was initiated.
Nuclear reconstitution procedures were utilized to determine the location of the deficiency in polyamine-depleted cell nuclei. It was shown that the nuclear salt extract from polyamine-depleted cells was slightly deficient, whereas the salt-extracted nuclear template could not support DNA synthesis.
Nuclear salt extracts (NSE's) were assayed for DNA polymerase alpha activity. Results showed that polyamine-depleted cell nuclei contained only $\sp\sim$50% as much DNA polymerase alpha activity found in control cell nuclei. This deficiency could also be reversed by the addition of spermidine to polyamine-depleted HeLa cells 10-12 hours before S-phase DNA synthesis was initiated.
Since the activity in NSE's obtained from polyamine-depleted cell nuclei reacted similarly with various DNA templates, it appears that a quantitative rather than a qualitative deficiency exists between the DNA polymerase alpha of control and polyamine-depleted HeLa cell nuclei.
Salt-extracted whole cell lysates (LSE's) were assayed for total DNA polymerase alpha activity. Results indicate that LSE's from polyamine-depleted cells contained levels of DNA polymerase alpha activity comparable to control cell LSE's. These results indicated that a transport problem may exist which prevents DNA polymerase alpha from entering, or allows DNA polymerase alpha to exit freely from the nucleus in polyamine-depleted cells.
In vitro nuclear protein phosphorylation studies revealed a 31 kdal protein that was not phosphorylated as control and polyamine-supplemented cells approached S-phase. However, polyamine-depleted HeLa cells continued to phosphorylate this protein in the comparable stage of the cell cycle.
Proliferation studies provided no evidence of the cells being synchronized via the DFMO block. This suggests that the polyamine deficiency does not block cells at a specific control site in the cell cycle. The cumulative evidence in this study indicates that polyamines are essential at several stages of cell cycle progression.
Koza, Robert Anthony, "Characterization of cell cycle changes in polyamine-depleted HeLa cells" (1989). Doctoral Dissertations. 1587.