Polyamines and HeLa-cell DNA replication

Abstract

HeLa cells were synchronized for S-phase DNA synthesis by the double thymidine-block procedure. A comparison was made of the polyamine content and S-phase DNA synthesis in cells from control cultures and cultures to which an inhibitor of polyamine biosynthesis, alpha-difluoromethylornithine, was added to the synchronization medium. Control cells showed a peak of synchronous DNA synthesis at 3 h and a maximum concentration of polyamines at 6-9 h after release of the second thymidine block. Cells from cultures containing the inhibitor were severely inhibited in the synthesis of DNA and contained no putrescine and only traces of spermidine while the spermine content was lowered by as much as 80%. Supplementation of cultures containing alpha-difluoromethylornithine with a polyamine, at the time of release of the second thymidine block, replenished the intracellular pool of the administered polyamine and partially restored S-phase DNA synthesis, with a lag of 3-6 h. Almost complete restoration of DNA synthesis in cells depleted of polyamines was achieved by the addition of a polyamine to cultures at least 10 h before release of the second thymidine block. The lag in initiation of synchronous S-phase DNA synthesis was eliminated in these cells. It is concluded that reversal by polyamines of the deficiency in S-phase DNA synthesis, in polyamine-depleted HeLa cells, is a time-dependent process indicative of the necessity for the replenishment of replication factors or their organization into an active replication complex.

Characterization of Acetate and Pyruvate Metabolism in Suspension Cultures of Zea mays by 13C NMR Spectroscopy

“…The subject of polyamines and their role in cellular process has been the subject of considerable work over the years. Recently, polyamines have been implicated as regulators of cell growth acting at the level of DNA replication and as modulators of membrane fusion (7,14). While it was not a goal here to evaluate the role of putrescine in corn suspension cells, one might speculate that a correlation may exist between the high intracellular concentration of putrescine observed here and the rapidly dividing nature ofthe corn suspension culture.…”

A Novel Intracellular Peptide Derived from G1/S Cyclin D2 Induces Cell Death

“…Therefore, we began by synchronizing HeLa cells using the double-thymidine block procedure (31,32) to further isolate and characterize intracellular peptides from each individual phase of the cell cycle. As a result of the double-thymidine block, analyses by flow cytometry suggested that ϳ84% of HeLa cells were arrested in each phase of the cell cycle (G 1 , S, or G 2 /M) when compared with the asynchronous cell group (Fig.…”

“…Cell Cycle Synchronization by Double-thymidine BlockHeLa cells were synchronized using the double-thymidine block procedure (31,32). Thymidine (Sigma-Aldrich) was diluted in serum-free DMEM and stored at 4°C before use.…”

Flow cytometric analysis of the cell cycle in polyamine‐depleted cells

“…The mechanism by which polyamine depletion affects cell growth, however, is not clear. Several studies seem to indicate that polyamines are required for the transition from G1-(GO) to S-phase (11,16,26, 28,291, but there are other reports suggesting that the progression through S-phase is most severely affected by polyamine depletion (4,6,18,31). CHO cells which are adapted to grow without serum and are polyamine dependent, due to an arginase deficiency (8), accumulate in the S-and G2-phases when grown without polyamines (1).…”