Growth state-dependent alterations in the ability of 12-O-tetradecanoylphorbol-13-acetate to increase ornithine decarboxylase activity in Reuber h35 Hepatoma cells.

Abstract

Ornithine decarboxylase (ODC) activity has been induced by tumor promoter 12- O -tetradecanoylphorbol-13-acetate (TPA) in rat hepatoma cells cultured under various medium conditions and growth states. A single application of 1.6 µm TPA was capable of inducing the ODC activity in 5- or 7-day-old cells maintained in the presence or absence of serum during promoter treatment. In the 5-day-old cells, TPA-mediated maximal ODC activity occurred by 3 hr and rapidly declined to control levels by 5 hr. The magnitudes of ODC inductions under serum-containing and serum-free conditions were comparable; however, due to variation in control levels of ODC at 0 hr, the relative increases in ODC activity were about 10-fold and 33-fold for cultures with and without serum, respectively. In the 7-day-old cells, the maximal activity of ODC occurred at 4 to 5 hr following TPA treatment. TPA caused a 100-fold increase in ODC activity in the 7-day cultures with serum and a 256-fold increase in those without serum. The ODC activity was slightly higher at 4 hr in the cells cultured with serum and remained at a high level for an additional 2 hr compared with cells cultured for 24 hr in the absence of serum. The effect of various concentrations of TPA upon the induction of ODC activity was dose dependent in the range of 1.6 × 10-8 m to 3.2 × 10-6 m. The 50% maximal increase in ODC activity was calculated to be 1.8 × 10-7 m and 4.3 × 10-7 m for cultures with and without serum, respectively. This suggests that the cells were more sensitive to TPA in the conditioned medium. Studies of TPA-stimulated ODC induction of cells at different growth states, i.e. , comparing log-phase cultures to confluent or stationary-phase cultures, indicated an increased responsiveness of cultures to TPA as the cultures reached 100% confluency. This increased responsiveness found in TPA-treated confluent cultures, however, did not occur when either dibutyryl cyclic adenosine 3′:5′-monophosphate or dexamethasone was used to induce ODC. In contrast, both agents appeared to be more effective in inducing ODC activity in the nonconfluent rapidly growing cultures. The intracellular concentrations of the polyamines were elevated following TPA treatment in cultures throughout the growth states, i.e. , from Day 3 to Day 8. In accord with ODC induction, putrescine concentration accumulated significantly within 3 hr, reached a maximal level by 6 hr, and then declined gradually to the basal level by 12 hr. While significant increase in spermidine concentration occurred at 8 hr, no changes in spermine concentration brought about by TPA occurred in any cultures studied. Our data also showed an apparent discrepancy between TPA-mediated maximal ODC induction and putrescine accumulation in cultures at different growth states. The maximal level to which ODC activity increased following incubation of the H35 cell cultures in various states with TPA was inversely proportional to the increase in intracellular putrescine. These data reinforce the suggestion that intracellular putrescine may play an important regulatory role in modulating ODC activity.