Date of Award

Fall 1980

Project Type

Dissertation

Program or Major

Biochemistry

Degree Name

Doctor of Philosophy

Abstract

Ornithine decarboxylase activity was stimulated in high-density HeLa cell cultures by dilution of or replacement of spent culture medium with fresh medium containing 10% (v/v) horse serum. After stimulation, ornithine decarboxylase activity reached a peak at 4-6 h. then rapidly declined to the low enzyme activity characteristic of quiescent cultures, where it remained during the remainder of the cell cycle. The stimulation of ornithine decarboxylase was eliminated by the addition of 0.5 (mu)M-spermine or spermidine or 10 (mu)M-putrescine to the HeLa cell cultures at the time of refeeding with fresh medium. Much higher concentrations (1 mM) of the non-physiological diamines, 1,3-diaminopropane or 1,3-diamino-2-hydroxy-propane, were required to eliminate the stimulation of ornithine decarboxylase in refed HeLa cell cultures. A heat labile, non-diffusible inhibitor, comparable with the inhibitory protein ornithine decarboxylase-antizyme, was induced in HeLa cells by the addition of exogenous diamines or polyamines. Intracellular putrescine was eliminated, intracellular spermidine and spermine were severely decreased and proliferation of HeLa cells was inhibited when cultures were maintained for 48 h. in the presence of the non-physiological inducer of ornithine decarboxylase-antizyme, 1,3-diamino-2-hydroxypropane. Exogenous putrescine, a physiological inducer of the antizyme, did not decrease intracellular polyamines or interfere with the proliferation of HeLa cells.

HeLa cells which were synchronized for DNA synthesis in the presence of 1,3-diamino-2-hydroxypropane (1.0 mM) and (alpha)-difluoromethylornithine (1.0 mM) exhibited a severe deficiency in S phase DNA synthesis and cell proliferation. This deficiency was reversible in that exogenous addition of polyamines at the beginning of S phase in the presence of (alpha)-difluoromethylornithine led to a restoration of the normal kinetics of S phase DNA synthesis and cell proliferation. The deficiency in DNA synthesis was also found in nuclei isolated from polyamine-depleted cells. Addition of spermidine in vitro to the isolated nuclei failed to restore the deficiency in DNA synthesis. Restoration of DNA synthesis in nuclei isolated from polyamine-depleted cells was accomplished by addition of 40 (mu)M spermidine to the cultures 28 h. prior to the isolation of nuclei.

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